Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/712,152, filed on Dec. 12, 2019, which is a continuation of U.S.patent application Ser. No. 15/782,091, filed on Oct. 12, 2017, now U.S.Pat. No. 10,570,393, issued on Feb. 25, 2020, which is a 35 § U.S.C.111(a) continuation application which claims the benefit of priority toPCT/US2016/027271, filed on Apr. 13, 2016, which in turn claims priorityto U.S. Provisional Application No. 62/146,604, filed on Apr. 13, 2015,and U.S. Provisional Application No. 62/261,361, filed on Dec. 1, 2015.The entire contents of each of the foregoing applications are herebyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 21, 2021, isnamed 121301_03305_SL.txt and is 213,367 bytes in size.

BACKGROUND OF THE INVENTION

Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-likefamily of secreted factors that regulates lipid metabolism and that ispredominantly expressed in the liver (Koishi, R. et al., (2002) Nat.Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activitiesof lipoprotein lipase (LPL), which catalyzes the hydrolysis oftriglycerides, and of endothelial lipase (EL), which hydrolyzes highdensity lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese,KK/Snk mice, a reduction in ANGPTL3 expression has a protective effectagainst hyperlipidemia and artherosclerosis by promoting the clearanceof triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223).Human ANGPTL3 plasma concentrations positively correlate with plasma HDLcholesterol and HDL phospholipid levels (Shimamura et al., (2007)Arterioscler. Thromb. Vasc. Biol., 27:366-372).

Disorders of lipid metabolism can lead to elevated levels of serumlipids, such as triglycerides and/or cholesterol. Elevated serum lipidsare strongly associated with high blood pressure, cardiovasculardisease, diabetes and other pathologic conditions. Hypertriglyceridemiais an example of a lipid metabolism disorder that is characterized byhigh blood levels of triglycerides. It has been associated withatherosclerosis, even in the absence of high cholesterol levels(hypercholesterolemia). When triglyceride concentrations are excessive(i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia canalso lead to pancreatitis. Hyperlipidemia is another example of a lipidmetabolism disorder that is characterized by elevated levels of any oneor all lipids and/or lipoproteins in the blood. Current treatments fordisorders of lipid metabolism, including dieting, exercise and treatmentwith statins and other drugs, are not always effective. Accordingly,there is a need in the art for alternative treatments for subjectshaving disorders of lipid metabolism.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using the iRNA compositions of the inventionfor inhibiting the expression of an ANGPL3 gene and/or for treating asubject who would benefit from inhibiting or reducing the expression ofan ANGPL3 gene, e.g., a subject suffering or prone to suffering from adisorder of lipid metabolism, such as a subject suffering or prone tosuffering from hyperlipidemia or hypertriglyceridemia.

Accordingly, in one aspect, the present invention providesdouble-stranded ribonucleic acids (dsRNAs) for inhibiting expression ofANGPTL3. The dsRNAs include a sense strand and an antisense strand,wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:1 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:5. In certain embodiments, the dsRNAs include asense strand and an antisense strand, wherein the sense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the antisense sequences listed in any one ofTables 2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14,15A, and 15B.

In another aspect, the invention provides double-stranded ribonucleicacids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs includea sense strand and an antisense strand, the antisense strand comprisinga region of complementarity which comprises at least 15 contiguousnucleotides differing no more than 3 nucleotides from any one of thenucleotide sequences selected from the group consisting of

(SEQ ID NO: 14) 5′-GAAUAUGUCACUUGAACUCAA-3′ (SEQ ID NO: 15)5′-UTGAGUUCAAGTGACAUAUUCUU-3′, (SEQ ID NO: 16)5′-GAAUATGUGACUUGAACUCAA-3′ (SEQ ID NO: 17)5′-UUGAGUUCAAGUGACAUAUUCUU-3′, (SEQ ID NO: 18)5′-AUUAAGCUGCUUCUUUTUAUU-3′ (SEQ ID NO: 19)5′-AAUAAAAAGAAGGAGCUUAAUUG-3′, (SEQ ID NO: 20)5′-ACAUAUUUGAUCAGUCUUUUU-3′ (SEQ ID NO: 21)5′-AAAAAGACUGAUCAAAUAUGUUG-3′; (SEQ ID NO: 22)5′-UGUCACUUGAACUCAACUCAA-3′ (SEQ ID NO: 23)5′-UUGAGUUGAGUUCAAGUGACAUA-3′; (SEQ ID NO: 24)5′-AACUAACUAACUUAAUUCAAA-3′ (SEQ ID NO: 25)5′-UUUGAAUUAAGUUAGUUAGUUGC-3′; (SEQ ID NO: 26)5′-UCACAAUUAAGCUCCUUCUUU-3′ (SEQ ID NO: 27);5′-AAAGAAGGAGCUUAAUUGUGAAC-3′ (SEQ ID NO: 28)5′-GAGCAACUAACUAACUUAAUU-3′ (SEQ ID NO: 29)5′-AAUUAAGUUAGUUAGUUGCUCUU-3′; (SEQ ID NO: 30)5′-UUAUUGUUCCUCUAGUUAUUU-3′ (SEQ ID NO: 31)5′-AAAUAACUAGAGGAACAAUAAAA-3′; (SEQ ID NO: 32)5′-AUUAAGCUCCUUCUUUUUAUU-3′ (SEQ ID NO: 33)5′-AAUAAAAAGAAGGAGCUUAAUUG-3′; (SEQ ID NO: 34)5′-GAAUAUGUCACUUGAACUCAA-3′ (SEQ ID NO: 35)5′-UUGAGUUCAAGUGACAUAUUCUU-3′; (SEQ ID NO: 36)5′-CAACAUAUUUGAUCAGUCUUU-3′ (SEQ ID NO: 37)5′-AAAGACUGAUCAAAUAUGUUGAG-3′; and (SEQ ID NO: 38)5′-CUCCAUAGUGAAGCAAUCUAA-3′ (SEQ ID NO: 39)5′-UUAGAUUGCUUCACUAUGGAGUA-3′.

In certain embodiments, the sense and antisense strands comprisenucleotide sequences selected from the group consisting of

(SEQ ID NO: 14) 5′-GAAUAUGUCACUUGAACUCAA-3′ (SEQ ID NO: 15)5′-UTGAGUUCAAGTGACAUAUUCUU-3′, (SEQ ID NO: 16)5′-GAAUATGUGACUUGAACUCAA-3′ (SEQ ID NO: 17)5′-UUGAGUUCAAGUGACAUAUUCUU-3′, (SEQ ID NO: 18)5′-AUUAAGCUGCUUCUUUTUAUU-3′ (SEQ ID NO: 19)5′-AAUAAAAAGAAGGAGCUUAAUUG-3′, (SEQ ID NO: 20)5′-ACAUAUUUGAUCAGUCUUUUU-3′ (SEQ ID NO: 21)5′-AAAAAGACUGAUCAAAUAUGUUG-3′; (SEQ ID NO: 22)5′-UGUCACUUGAACUCAACUCAA-3′ (SEQ ID NO: 23)5′-UUGAGUUGAGUUCAAGUGACAUA-3′; (SEQ ID NO: 24)5′-AACUAACUAACUUAAUUCAAA-3′  (SEQ ID NO: 25)5′-UUUGAAUUAAGUUAGUUAGUUGC-3′; (SEQ ID NO: 26)5′-UCACAAUUAAGCUCCUUCUUU-3′  (SEQ ID NO: 27)5′-AAAGAAGGAGCUUAAUUGUGAAC-3′; (SEQ ID NO: 28)5′-GAGCAACUAACUAACUUAAUU-3′  (SEQ ID NO: 29)5′-AAUUAAGUUAGUUAGUUGCUCUU-3′; (SEQ ID NO: 30)5′-UUAUUGUUCCUCUAGUUAUUU-3′ (SEQ ID NO: 31)5′-AAAUAACUAGAGGAACAAUAAAA-3′; (SEQ ID NO: 32)5′-AUUAAGCUCCUUCUUUUUAUU-3′ (SEQ ID NO: 33)5′-AAUAAAAAGAAGGAGCUUAAUUG-3′; (SEQ ID NO: 34)5′-GAAUAUGUCACUUGAACUCAA-3′ (SEQ ID NO: 35)5′-UUGAGUUCAAGUGACAUAUUCUU-3′; (SEQ ID NO: 36)5′-CAACAUAUUUGAUCAGUCUUU-3′ (SEQ ID NO: 37)5′-AAAGACUGAUCAAAUAUGUUGAG-3′; and (SEQ ID NO: 38)5′-CUCCAUAGUGAAGCAAUCUAA-3′ (SEQ ID NO: 39)5′-UUAGAUUGCUUCACUAUGGAGUA-3′.

In certain embodiments, the dsRNA comprises at least one modifiednucleotide. In certain embodiments, the dsRNA comprises no more than 4(i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the sense strand. Incertain embodiments, the dsRNA comprises no more than 4 (i.e., 4, 3, 2,1, or 0) unmodified nucleotides in the antisense strand. In certainembodiments, the dsRNA comprises no more than 4 (i.e., 4, 3, 2, 1, or 0)unmodified nucleotides in both the sense strand and the antisensestrand. In certain embodiments, all of the nucleotides in the sensestrand of the dsRNA are modified nucleotides. In certain embodiments,all of the nucleotides in the antisense strand of the dsRNA are modifiednucleotides. In certain embodiments, all of the nucleotides in the sensestrand of the dsRNA and all of the nucleotides of the antisense strandare modified nucleotides.

In certain embodiments, the modified nucleotides is/are independentlyselected from the group consisting of a 2′-O-methyl modified nucleotide,a nucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or a dodecanoic acidbisdecylamide group. In certain embodiments, the modified nucleotide isselected from the group consisting of a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modifiednucleotide, a morpholino nucleotide, a phosphoramidate, and anon-natural base comprising nucleotide.

In certain embodiments, the dsRNA comprises a region of complementarityat least 17 nucleotides in length. In certain embodiments, the dsRNAcomprises a region of complementarity 19 and 23 nucleotides in length.In certain embodiments, the dsRNA comprises a region of complementarityis 19 nucleotides in length.

In certain embodiments, each strand of the dsRNA is no more than 30nucleotides in length. In certain embodiments, the dsRNA is at least 15nucleotides in length.

In other embodiments, one or both of the strands of the double strandedRNAi agents of the invention is up to 66 nucleotides in length, e.g.,36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with aregion of at least 19 contiguous nucleotides that is substantiallycomplementary to at least a part of an mRNA transcript of an INSR gene.In some embodiments, the sense and antisense strands form a duplex of18-30 contiguous nucleotides.

In one embodiment, at least one strand of the RNAi agent comprises a 3′overhang of at least 1 nucleotide. In certain embodiments, at least onestrand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4,5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments,at least one strand of the RNAi agent comprises a 5′ overhang of atleast 1 nucleotide. In certain embodiments, at least one strandcomprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments,both the 3′ and the 5′ end of one strand of the RNAi agent comprise anoverhang of at least 1 nucleotide.

In certain embodiments, the double stranded RNAi agent further comprisesa ligand. In certain embodiments, the ligand is an N-acetylgalactosamine(GalNAc). The ligand may be one or more GalNAc attached to the RNAiagent through a monovalent, a bivalent, or a trivalent branched linker.The ligand may be conjugated to the 3′ end of the sense strand of thedouble stranded RNAi agent, the 5′ end of the sense strand of the doublestranded RNAi agent, the 3′ end of the antisense strand of the doublestranded RNAi agent, or the 5′ end of the antisense strand of the doublestranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the inventioncomprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, eachindependently attached to a plurality of nucleotides of the doublestranded RNAi agent through a plurality of monovalent linkers.

In certain embodiments, the ligand is

In certain embodiments, the double stranded RNAi agent is conjugated tothe ligand as shown in the following schematic

and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the dsRNA comprises a duplex selected from thegroup consisting of AD-57927.6, AD-63133.1, AD-63136.1, AD-63137.1,AD-63139.1, AD-63142.1, AD-63143.1, AD-63144.1, AD-63145.1, AD-63148.1,AD-63149.1, AD-63150.1, AD-63151.1, AD-63153.1, AD-63154.1, AD-63156.1,AD-63157.1, AD-63160.1, AD-63162.1, AD-63163.1, AD-63167.1, AD-63168.1,AD-63170.1, AD-63173.1, AD-63174.1, AD-63175.1, AD-63176.1, AD-63177.1,AD-63179.1, AD-63181.1, AD-66916, AD-66920, AD-66921, AD-66923,AD-66922, AD-66917, AD-66918, AD-66919, AD-66924, AD66925 andAD-63185.1. In certain embodiments, the dsRNA comprises a duplexselected from the group consisting of AD-57927.6, AD-63136.1,AD-63137.1, AD-63142.1, D-63148.1, AD-63151.1, AD-63156.1, AD-63157.1,AD-63160.1, AD-63163.1, AD-63167.1, AD-63170.1, AD-63173.1, AD-63174.1,AD-63176.1, AD-66916, AD-66920, AD-66921, AD-66923, AD-66922, AD-66917,AD-66918, AD-66919, AD-66924, AD66925, AD-67173, AD-67174, AD-67007, andAD-63179.1. In one embodiment, the sense and antisense strands of thedsRNA comprise nucleotide sequences selected from the group consistingof

(SEQ ID NO: 40) 5′-ascsauauUfuGfAfUfcagucuuuuu-3′ (SEQ ID NO: 41)5′-asAfsaaaGfacugaucAfaAfuaugususg-3′; (SEQ ID NO: 42)5′-usgsucacUfuGfAfAfcucaacucaaL96-3′ (SEQ ID NO: 43)5′-usUfsgagUfuGfAfguucAfaGfugacasusa-3′; (SEQ ID NO: 44)5′-gsasauauGfuCfAfCfuugaacucaa-3′ (SEQ ID NO: 45)5′-usdTsgaguucaagdTgdAcauauucsus-3′; (SEQ ID NO: 46)5′-gsasauadTgudGacuugaa(Cgn)ucaa-3′ (SEQ ID NO: 47)5′-usUfsgagUfuCfAfagugAfcAfuauucsusu-3′; (SEQ ID NO: 48)5′-asusuaadGcudGcuucuuu(Tgn)uauu-3′ (SEQ ID NO: 49)5′-asAfsuaaAfaagaaggAfgCfuuaaususg-3′; (SEQ ID NO: 50)5′-AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf-3′ (SEQ ID NO: 51)5′-asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg-3′; (SEQ ID NO: 52)5′-UfsgsUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf-3′ (SEQ ID NO: 53)5′-usUfsgAfgUfuGfaGfuucAfaGfuGfaCfasusa-3′; (SEQ ID NO: 54)5′-asascuaaCfuAfAfCfuuaauucaaa-3′, (SEQ ID NO: 55)5′-usUfsugaAfuUfAfaguuAfgUfuaguusgsc-3′; (SEQ ID NO: 56)5′-uscsacaaUfuAfAfGfcuccuucuuu-3′ (SEQ ID NO: 57)5′-asAfsagaAfgGfAfgcuuAfaUfugugasasc-3′; (SEQ ID NO: 58)5′-gsasgcaaCfuAfAfCfuaacuuaauu-3′ (SEQ ID NO: 59)5′-asAfsuuaAfgUfUfaguuaGfuUfgcucsusu-3′; (SEQ ID NO: 60)5′-ususauugUfuCfCfUfcuaguuauuu-3′ (SEQ ID NO: 61)5′-asAfsauaAfcUfAfgaggAfaCfaauaasasa-3′; (SEQ ID NO: 62)5′-asusuaagCfuCfCfUfucuuuuuauu-3′ (SEQ ID NO: 63)5′-asAfsuaaAfaAfGfaaggAfgCfuuaaususg-3′; (SEQ ID NO: 64)5′-gsasauauGfuCfAfCfuugaacucaa-3′ (SEQ ID NO: 65)5′-usUfsgagUfuCfAfagugAfcAfuauucsusu-3′; (SEQ ID NO: 66)5′-csasacauAfuUfUfGfaucagucuuu-3′ (SEQ ID NO: 67)5′-asAfsagaCfuGfAfucaaAfuAfuguugsasg-3′; and (SEQ ID NO: 68)5′-csusccauAfgUfGfAfagcaaucuaa-3′ (SEQ ID NO: 69)5′-usUfsagaUfuGfCfuucaCfuAfuggagsusa-3′.

In another aspect, the invention provides cells containing the dsRNAsprovided herein.

In yet another aspect, the invention provides pharmaceuticalcompositions for inhibiting expression of an ANGPTL3 gene. Thecompositions include the dsRNA agents provided herein. In certainembodiments, the pharmaceutical compositions further comprise a lipidformulation.

The invention also provides methods of inhibiting ANGPTL3 expression ina cell. The methods include contacting the cell with the dsRNA asdescribed herein; and maintaining the cell for a time sufficient toobtain degradation of the mRNA transcript of an ANGPTL3 gene, therebyinhibiting expression of the ANGPTL3 gene in the cell.

In certain embodiments, the cell is within a subject. In certainembodiments, the subject is a human. In certain embodiments, the humansubject suffers from a disorder of lipid metabolism. In certainembodiments, the disorder of lipid metabolism is hyperlipidemia orhypertriglyceridemia. In certain embodiments, the ANGPTL3 expression isinhibited by at least about 30%.

In another aspect, the invention provides methods of treating a subjecthaving a disorder that would benefit from reduction in ANGPTL3expression. The methods include administering to the subject atherapeutically effective amount of any of the dsRNAs provided herein,thereby treating the subject. In certain embodiments, the disorder is adisorder of lipid metabolism. In certain embodiments, the disorder oflipid metabolism is hyperlipidemia or hypertriglyceridemia. In certainembodiments, administration of the dsRNA to the subject causes adecrease in one or more serum lipid and/or a decrease in ANGPTL3 proteinaccumulation.

In a further aspect, the present invention also provides methods ofinhibiting the expression of ANGPTL3 in a subject. The methods includeadministering to the subject a therapeutically effective amount of anyof the dsRNAs provided herein, thereby inhibiting the expression ofANGPTL3 in the subject.

In yet another aspect, the invention provides kits for performing themethods of the invention. In one embodiment, the invention provides akit for performing a method of inhibiting expression of ANGPTL3 gene ina cell by contacting a cell with a double stranded RNAi agent of theinvention in an amount effective to inhibit expression of the ANGPTL3 inthe cell. The kit comprises an RNAi agent and instructions for use and,optionally, means for administering the RNAi agent to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the amount of ANGPTL3 mRNA remaining at day 3in the serum of ob/ob female mice after a single 30 mg/kg, 10 mg/kg, or3 mg/kg subcutaneous dose of the indicated iRNA agents. The amount ofANGPTL3 mRNA is presented as a ratio of the amount of ANGPTL3 mRNA tothe amount GAPDH mRNA in a sample relative to the ratio of the amount ofANGPTL3 mRNA to the amount GAPDH mRNA in a control sample (PBS).

FIG. 2 is a graph showing the effects of a multi-dose administration (3mg/kg every day for 5 days during week 1, followed by 3 mg/kg two timesper week on weeks 2-4 (3.0 mg/kg qd×5; qw×6)) of AD-57927 on serumANGPTL3 protein levels in ob/ob female mice. The amount of ANGPTL3protein presented is relative to the amount ANGPTL3 protein present in aserum sample prior to administration of AD-57927.

FIG. 3 is a graph showing the effects of a multi-dose administration (3mg/kg every day for 5 days during week 1, followed by 3 mg/kg two timesper week on weeks 2-4 (3.0 mg/kg qd×5; qw×6)) of AD-57927 ontriglyceride (TG) levels in ob/ob female mice. The amount of TGpresented is relative to the amount TG present in a serum sample priorto administration of AD-57927.

FIG. 4 is a graph showing the effects of a multi-dose administration (3mg/kg every day for 5 days during week 1, followed by 3 mg/kg two timesper week on weeks 2-4 (3.0 mg/kg qd×5; qw×6)) of AD-57927 on LDLcholesterol (LDLc) levels in ob/ob female mice. The amount of LDLcpresented is relative to the amount LDLc present in a serum sample priorto administration of AD-57927.

FIG. 5 is a graph showing the effects of a multi-dose administration (3mg/kg every day for 5 days during week 1, followed by 3 mg/kg two timesper week on weeks 2-4 (3.0 mg/kg qd×5; qw×6)) of AD-57927 on totalcholesterol (Tc) levels in ob/ob female mice. The amount of Tc presentedis relative to the amount Tc present in a serum sample prior toadministration of AD-57927.

FIG. 6A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of wild-type mice after a single 3 mg/kg subcutaneous doseof the indicated iRNA agents over time. The amount of mouse ANGPTL3protein presented is relative to the amount mouse ANGPTL3 proteinpresent in a serum sample prior to administration.

FIG. 6B is a graph showing the amount of mouse ANGPTL3 protein remainingin the serum of wild-type mice after a single 3 mg/kg subcutaneous doseof the indicated iRNA agents on day 10 post-dose. The amount of mouseANGPTL3 protein presented is relative to the amount mouse ANGPTL3protein present in a serum sample prior to administration.

FIG. 7A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of wild-type mice after a single 3 mg/kg subcutaneous doseof the indicated iRNA agents over time. The amount of mouse ANGPTL3protein presented is relative to the amount mouse ANGPTL3 proteinpresent in a serum sample prior to administration.

FIG. 7B is a graph showing the amount of mouse ANGPTL3 protein remainingin the serum of wild-type mice after a single 1 mg/kg or 3 mg/kgsubcutaneous dose of the indicated iRNA agents on day 5 post-dose. Theamount of mouse ANGPTL3 protein presented is relative to the amountmouse ANGPTL3 protein present in a serum sample prior to administration.

FIG. 8A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of ob/ob mice after a single 3 mg/kg subcutaneous dose ofthe indicated iRNA agents over time. The amount of mouse ANGPTL3 proteinpresented is relative to the amount mouse ANGPTL3 protein present in aserum sample prior to administration.

FIG. 8B is a graph showing the percent of silencing of mouse ANGPTL3protein in the serum of ob/ob mice after a single 1 mg/kg or 3 mg/kgsubcutaneous dose of the indicated iRNA agents on day 5 post-dose. Thepercent of silencing of mouse ANGPTL3 protein presented is relative tothe level of mouse ANGPTL3 protein present in a serum sample prior toadministration.

FIG. 9 is a graph showing the percent of silencing of mouse ANGPTL3protein in the serum of ob/ob mice after a single 0.3 mg/kg, 1 mg/kg, 3mg/kg, or 9 mg/kg subcutaneous dose of AD-65695 on day 5 post-dose. Thepercent of silencing of mouse ANGPTL3 protein is relative to the levelof mouse ANGPTL3 protein present in a serum sample prior toadministration.

FIG. 10A is a graph showing the effects of a single 0.3 mg/kg, 1 mg/kg,3 mg/kg, or 9 mg/kg subcutaneous dose of AD-65695 (ALN-ANG) ontriglyceride (TG) levels in ob/ob mice. The amount of TG is presented asa ratio of the amount of TG in a serum sample from a mouse administeredAD-65695 relative to the amount of TG in a serum sample from a mouseadministered PBS.

FIG. 10B is a graph showing the effects of a single 0.3 mg/kg, 1 mg/kg,3 mg/kg, or 9 mg/kg subcutaneous dose of AD-65695 (ALN-ANG) on totalcholesterol (TC) levels in ob/ob mice. The amount of TC is presented asa ratio of the amount of TC in a serum sample from a mouse administeredAD-65695 relative to the amount of TC in a serum sample from a mouseadministered PBS.

FIG. 10C is a graph showing the effects of a single 0.3 mg/kg, 1 mg/kg,3 mg/kg, or 9 mg/kg subcutaneous dose of AD-65695 (ALN-ANG) on lowdensity lipoprotein cholesterol (LDLc) levels in ob/ob mice. The amountof LDLc is presented as a ratio of the amount of LDLc in a serum samplefrom a mouse administered AD-65695 relative to the amount of LDLc in aserum sample from a mouse administered PBS.

FIG. 10D is a graph showing the effects of a single 0.3 mg/kg, 1 mg/kg,3 mg/kg, or 9 mg/kg subcutaneous dose of AD-65695 (ALN-ANG) on the ratioof high density lipoprotein cholesterol (HDL) levels to totalcholesterol (TC) levels in ob/ob mice.

FIG. 11 depicts the dosing schedule and study design for determining thedurability of the indicated iRNA agents in wild-type mice (C57BL/6)infected by intravenous administration of 1×10¹¹ viral particles of anadeno-associated virus 8 (AAV8) vector encoding the human ANGPTL3 gene(coding region) driven by the liver-specific thyroxine-binding globulin(TBG) promoter (AAV8-TBG-ANGPTL3).

FIG. 12A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of human ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 3 mg/kgsubcutaneous dose of the indicated iRNA agents over time. The amount ofhuman ANGPTL3 protein presented is relative to the amount of humanANGPTL3 protein present in a serum sample prior to administration(pre-dose).

FIG. 12B is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 3 mg/kgsubcutaneous dose of the indicated iRNA agents over time. The amount ofmouse ANGPTL3 protein presented is relative to the amount of mouseANGPTL3 protein present in a serum sample prior to administration(pre-dose).

FIG. 13 depicts the dosing schedule and study design for titrating thedose and determining the durability of the indicated iRNA agents inAAV8-TBG-ANGPTL3 infected mice.

FIG. 14A is a graph showing the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single3 mg/kg, 1 mg/kg, or 0.3 mg/kg subcutaneous dose of the indicated iRNAagents at day 11 post-dose. The amount of human ANGPTL3 proteinpresented is relative to the amount of human ANGPTL3 protein present ina serum sample prior to administration (pre-dose).

FIG. 14B is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 3 mg/kg, 1mg/kg, or 0.3 mg/kg subcutaneous dose of the indicated iRNA agents atday 11 post-dose. The amount of mouse ANGPTL3 protein presented isrelative to the amount of mouse ANGPTL3 protein present in a serumsample prior to administration (pre-dose).

FIG. 15A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of human ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 3 mg/kg, 1mg/kg, or 0.3 mg/kg subcutaneous dose of the indicated iRNA agents overtime. The amount of human ANGPTL3 protein presented is relative to theamount of human ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

FIG. 15B is a graph showing the duration of response to the indicatediRNA agents represented by the amount of mouse ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 3 mg/kg, 1mg/kg, or 0.3 mg/kg subcutaneous dose of the indicated iRNA agents overtime. The amount of mouse ANGPTL3 protein presented is relative to theamount of mouse ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

FIG. 16 is a graph showing the duration of response to the indicatediRNA agents represented by the amount of human ANGPTL3 protein remainingin the serum of AAV8-TBG-ANGPTL3 infected mice after a single 1 mg/kgsubcutaneous dose of the indicated iRNA agents over time. The amount ofhuman ANGPTL3 protein presented is relative to the amount of humanANGPTL3 protein present in a serum sample prior to administration(pre-dose).

FIG. 17A is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg or 3 mg/kg subcutaneous dose of the indicated iRNA agents at day14 post-dose. The amount of human ANGPTL3 protein presented is relativeto the amount of human ANGPTL3 protein present in a serum sample priorto administration (pre-dose).

FIG. 17B is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg or 3 mg/kg subcutaneous dose of the indicated iRNA agents at day28 post-dose. The amount of human ANGPTL3 protein presented is relativeto the amount of human ANGPTL3 protein present in a serum sample priorto administration (pre-dose).

FIG. 18A is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg or 3 mg/kg subcutaneous dose of the indicated iRNA agents at day14 post-dose. The amount of human ANGPTL3 protein presented is relativeto the amount of human ANGPTL3 protein present in a serum sample priorto administration (pre-dose).

FIG. 18B is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg or 3 mg/kg subcutaneous dose of the indicated iRNA agents at day28 post-dose. The amount of human ANGPTL3 protein presented is relativeto the amount of human ANGPTL3 protein present in a serum sample priorto administration (pre-dose).

FIG. 19A is a graph showing the duration of response to the indicatediRNA agents represented by the amount of Cynomolgus ANGPTL3 proteinremaining in the serum of lean Cynomolgus monkeys administered a single1 mg/kg or 3 mg/kg dose of the indicated iRNA agents over time. Theamount of Cynomolgus ANGPTL3 protein presented is relative to the amountof human ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

FIG. 19B is a graph showing the percent of triglycerides remaining inthe serum of Cynomolgus monkeys administered a single 1 mg/kg or 3 mg/kgdose of the indicated iRNA agents over time. The amount of triglyceridespresented is relative to the amount of Cynomolgus triglycerides presentin a serum sample prior to administration (pre-dose).

FIG. 20 is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg subcutaneous dose of the indicated iRNA agents at day 28post-dose. The amount of human ANGPTL3 protein presented is relative tothe amount of human ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

FIG. 21A is a graph depicting the amount of human ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg subcutaneous dose of the indicated iRNA agents at day 28post-dose. The amount of human ANGPTL3 protein presented is relative tothe amount of human ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

FIG. 21B is a graph depicting the amount of mouse ANGPTL3 proteinremaining in the serum of AAV8-TBG-ANGPTL3 infected mice after a single1 mg/kg subcutaneous dose of the indicated iRNA agents at day 28post-dose. The amount of human ANGPTL3 protein presented is relative tothe amount of human ANGPTL3 protein present in a serum sample prior toadministration (pre-dose).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using the iRNA compositions of the inventionfor inhibiting the expression of an ANGPTL3 gene and/or for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an ANGPTL3 gene, e.g., a disorder of lipid metabolism,such as hyperlipidemia or hypertriglyceridemia.

The iRNAs of the invention may include an RNA strand (the antisensestrand) having a region which is about 30 nucleotides or less in length,e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22,15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26,18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27,19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28,20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28,21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, whichregion is substantially complementary to at least part of an mRNAtranscript of an ANGPTL3 gene.

In other embodiments, one or both of the strands of the double strandedRNAi agents of the invention is up to 66 nucleotides in length, e.g.,36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with aregion of at least 19 contiguous nucleotides that is substantiallycomplementary to at least a part of an mRNA transcript of an ANGPTL3gene. In some embodiments, the sense and antisense strands form a duplexof 18-30 contiguous nucleotides.

The use of these iRNA agents described herein enables the targeteddegradation of mRNAs of an ANGPTL3 gene in mammals. Very low dosages ofANGPTL3 iRNAs, in particular, can specifically and efficiently mediateRNA interference (RNAi), resulting in significant inhibition ofexpression of an ANGPTL3 gene. Using cell-based and in vivo assays, thepresent inventors have demonstrated that iRNAs targeting ANGPTL3 canmediate RNAi, resulting in significant inhibition of expression of anANGPTL3 gene. Thus, methods and compositions including these iRNAs areuseful for treating a subject who would benefit by a reduction in thelevels and/or activity of an ANGPTL3 protein, such as a subject having adisorder of lipid metabolism, such as hyperlipidemia orhypertriglyceridemia.

In some embodiments, the iRNA agents of the invention include an RNAstrand (the antisense strand) which can be up to 66 nucleotides inlength, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides inlength, with a region of at least 19 contiguous nucleotides that issubstantially complementary to at least a part of an mRNA transcript ofan ANGPTL3 gene. In some embodiments, such iRNA agents having longerlength antisense strands may include a second RNA strand (the sensestrand) of 20-60 nucleotides in length wherein the sense and antisensestrands form a duplex of 18-30 contiguous nucleotides.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of an ANGPTL3gene, as well as compositions and methods for treating subjects havingdiseases and disorders that would benefit from inhibition and/orreduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”. The term “or” is usedherein to mean, and is used interchangeably with, the term “and/or,”unless context clearly indicates otherwise.

The term “ANGPTL3” refers to an angiopoietin like protein 3 having anamino acid sequence from any vertebrate or mammalian source, including,but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine,ovine, primate, monkey, and guinea pig, unless specified otherwise. Theterm also refers to fragments and variants of native ANGPTL3 thatmaintain at least one in vivo or in vitro activity of a native ANGPTL3.The term encompasses full-length unprocessed precursor forms of ANGPTL3as well as mature forms resulting from post-translational cleavage ofthe signal peptide and forms resulting from proteolytic processing ofthe fibrinogen-like domain. The sequence of a human ANGPTL3 mRNAtranscript can be found at, for example, GenBank Accession No. GI:452408443 (NM_014495.3; SEQ ID NO:1). The predicted sequence of rhesusANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI:297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3mRNA can be found at, for example, GenBank Accession No. GI: 142388354(NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can befound at, for example, GenBank Accession No. GI: 68163568(NM_001025065.1; SEQ ID NO:4). Additional examples of ANGPTL3 mRNAsequences are readily available using publicly available databases,e.g., GenBank, UniProt, and OMIM.

The term“ANGPTL3” as used herein also refers to a particular polypeptideexpressed in a cell by naturally occurring DNA sequence variations ofthe ANGPTL3 gene, such as a single nucleotide polymorphism in theANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identifiedand may be found at, for example, NCBI dbSNP (see, e.g.,www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within theANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039;rs192778191; rs192764027; rs192528948; rs191931953; rs191293319;rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an ANGPTL3 gene, including mRNA that is a product of RNA processingof a primary transcription product. In one embodiment, the targetportion of the sequence will be at least long enough to serve as asubstrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an ANGPTL3gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 1). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of ANGPTL3 in a cell, e.g., a cell within a subject, suchas a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a ANGPTL3target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (sssiRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a ANGPTL3 gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNAiagent that is introduced into a cell or organism to inhibit a targetmRNA. Single-stranded RNAi agents (ssRNAi) bind to the RISCendonuclease, Argonaute 2, which then cleaves the target mRNA. Thesingle-stranded siRNAs are generally 15-30 nucleotides and arechemically modified. The design and testing of single-stranded RNAiagents are described in U.S. Pat. No. 8,101,348 and in Lima et al.,(2012) Cell 150: 883-894, the entire contents of each of which arehereby incorporated herein by reference. Any of the antisense nucleotidesequences described herein may be used as a single-stranded siRNA asdescribed herein or as chemically modified by the methods described inLima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions and methodsof the invention is a double-stranded RNA and is referred to herein as a“double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,”“dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex ofribonucleic acid molecules, having a duplex structure comprising twoanti-parallel and substantially complementary nucleic acid strands,referred to as having “sense” and “antisense” orientations with respectto a target RNA, i.e., an ANGPTL3 gene. In some embodiments of theinvention, a double-stranded RNA (dsRNA) triggers the degradation of atarget RNA, e.g., an mRNA, through a post-transcriptional gene-silencingmechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/or amodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to directthe cleavage of the target RNA. Without wishing to be bound by theory,long double stranded RNA introduced into cells is broken down into siRNAby a Type III endonuclease known as Dicer (Sharp et al. (2001) GenesDev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNAinto 19-23 base pair short interfering RNAs with characteristic two base3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or theantisense strand, or both, can include extended lengths longer than 10nucleotides, e.g., 10-30 nucleotides, 10-25 nucleotides, 10-20nucleotides or 10-15 nucleotides in length. In certain embodiments, anextended overhang is on the sense strand of the duplex. In certainembodiments, an extended overhang is present on the 3′end of the sensestrand of the duplex. In certain embodiments, an extended overhang ispresent on the 5′end of the sense strand of the duplex. In certainembodiments, an extended overhang is on the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the3′end of the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 5′end of the antisense strand of theduplex. In certain embodiments, one or more of the nucleotides in theextended overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an ANGPTL3 mRNA. As usedherein, the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an ANGPTL3 nucleotide sequence, asdefined herein. Where the region of complementarity is not fullycomplementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotideis complementary to at least a part of an ANGPTL3mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding ANGPTL3.

Accordingly, in some embodiments, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target ANGPTL3 sequence.In other embodiments, the antisense strand polynucleotides disclosedherein are substantially complementary to the target ANGPTL3 sequenceand comprise a contiguous nucleotide sequence which is at least about80% complementary over its entire length to the equivalent region of thenucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, suchas about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target ANGPTL3sequence, and wherein the sense strand polynucleotide comprises acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of the nucleotidesequence of SEQ ID NO:5, or a fragment of any one of SEQ ID NO:5, suchas about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary. The term “inhibiting,”as used herein, is used interchangeably with “reducing,” “silencing,”“downregulating,” “suppressing” and other similar terms, and includesany level of inhibition.

The phrase “inhibiting expression of an ANGPTL3,” as used herein,includes inhibition of expression of any ANGPTL3 gene (such as, e.g., amouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or ahuman ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 genethat encode an ANGPTL3 protein.

“Inhibiting expression of an ANGPTL3 gene” includes any level ofinhibition of an ANGPTL3 gene, e.g., at least partial suppression of theexpression of an ANGPTL3 gene, such as an inhibition by at least about20%. In certain embodiments, inhibition is by at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%.

The expression of an ANGPTL3 gene may be assessed based on the level ofany variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNAlevel or ANGPTL3 protein level. The expression of an ANGPTL3 may also beassessed indirectly based on the levels of a serum lipid, atriglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C andtotal cholesterol), or free fatty acids. Inhibition may be assessed by adecrease in an absolute or relative level of one or more of thesevariables compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of anANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNAwhich can be isolated from or detected in a first cell or group of cellsin which an ANGPTL3 gene is transcribed and which has or have beentreated such that the expression of an ANGPTL3 gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has or have not been sotreated (control cells). The degree of inhibition may be expressed interms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in ANGPTL3 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inANGPTL3 expression; a human having a disease, disorder or condition thatwould benefit from reduction in ANGPTL3 expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in ANGPTL3 expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, such as lowering levels oftriglycerides in a subject. The terms “treating” or “treatment” alsoinclude, but are not limited to, alleviation or amelioration of one ormore symptoms of a disorder of lipid metabolism, such as, e.g., adecrease in the size of eruptive xanthomas. “Treatment” can also meanprolonging survival as compared to expected survival in the absence oftreatment.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an ANGPTL3 gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with suchdisease, disorder, or condition, e.g., high triglyceride levels oreruptive xanthoma. The likelihood of developing a high tryglyceridelevels or eruptive xanthoma is reduced, for example, when an individualhaving one or more risk factors for a high tryglyceride levels oreruptive xanthoma either fails to develop high tryglyceride levels oreruptive xanthoma or develops high tryglyceride levels or eruptivexanthoma with less severity relative to a population having the samerisk factors and not receiving treatment as described herein. Thefailure to develop a disease, disorder or condition, or the reduction inthe development of a symptom associated with such a disease, disorder orcondition i (e.g., by at least about 10% on a clinically accepted scalefor that disease or disorder), or the exhibition of delayed symptomsdelayed (e.g., by days, weeks, months or years) is considered effectiveprevention.

As used herein, the term “serum lipid” refers to any major lipid presentin the blood. Serum lipids may be present in the blood either in freeform or as a part of a protein complex, e.g., a lipoprotein complex.Non-limiting examples of serum lipids may include triglycerides andcholesterol, such as total cholesterol (TG), low density lipoproteincholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), verylow density lipoprotein cholesterol (VLDL-C) and intermediate-densitylipoprotein cholesterol (IDL-C).

As used herein, a “disorder of lipid metabolism” refers to any disorderassociated with or caused by a disturbance in lipid metabolism. Forexample, this term includes any disorder, disease or condition that canlead to hyperlipidemia, or condition characterized by abnormal elevationof levels of any or all lipids and/or lipoproteins in the blood. Thisterm refers to an inherited disorder, such as familialhypertriglyceridemia, familial partial lipodystrophy type 1 (FPLD1), oran induced or acquired disorder, such as a disorder induced or acquiredas a result of a disease, disorder or condition (e.g., renal failure), adiet, or intake of certain drugs (e.g., as a result of highly activeantiretroviral therapy (HAART) used for treating, e.g., AIDS or HIV).Exemplary disorders of lipid metabolism include, but are not limited to,atherosclerosis, dyslipidemia, hypertriglyceridemia (includingdrug-induced hypertriglyceridemia, diuretic-inducedhypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergicblocking agent-induced hypertriglyceridemia, estrogen-inducedhypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia,retinoid-induced hypertriglyceridemia, cimetidine-inducedhypertriglyceridemia, and familial hypertriglyceridemia), acutepancreatitis associated with hypertriglyceridemia, chylomicron syndrom,familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiencyor hypoactivity, hyperlipidemia (including familial combinedhyperlipidemia), hypercholesterolemia, gout associated withhypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits),hyperlipidemia with heterogeneous LPL deficiency, and hyperlipidemiawith high LDL and heterogeneous LPL deficiency.

Cardiovascular diseases associated with disorders of lipid metabolismare also considered “disorders of lipid metabolism”, as defined herein.These diseases may include coronary artery disease (also called ischemicheart disease), inflammation associated with coronary artery disease,restenosis, peripheral vascular diseases, and stroke.

Disorders related to body weight are also considered “disorders of lipidmetabolism”, as defined herein. Such disorders may include obesity,metabolic syndrome including independent components of metabolicsyndrome (e.g., central obesity, FBG/pre-diabetes/diabetes,hypercholesterolemia, hypertriglyceridemia, and hypertension),hypothyroidism, uremia, and other conditions associated with weight gain(including rapid weight gain), weight loss, maintenance of weight loss,or risk of weight regain following weight loss.

Blood sugar disorders are further considered “disorders of lipidmetabolism”, as defined herein. Such disorders may include diabetes,hypertension, and polycystic ovarian syndrome related to insulinresistance. Other exemplary disorders of lipid metabolism may alsoinclude renal transplantation, nephrotic syndrome, Cushing's syndrome,acromegaly, systemic lupus erythematosus, dysglobulinemia,lipodystrophy, glycogenosis type I, and Addison's disease.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving a disorder of lipid metabolism, is sufficient to effect treatmentof the disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subjecthaving a disorder of lipid metabolism, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the iRNA, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to blood or plasma drawn from the subject.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of an ANGPTL3gene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having a disorder of lipid metabolism, e.g., familialhyperlipidemia. The dsRNA includes an antisense strand having a regionof complementarity which is complementary to at least a part of an mRNAformed in the expression of an ANGPTL3 gene, The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the ANGPTL3 gene,the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, aprimate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of anANGPTL3gene. The other strand (the sense strand) includes a region thatis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 23nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well known in the art that dsRNAslonger than about 21-23 nucleotides can serve as substrates for Dicer.As the ordinarily skilled person will also recognize, the region of anRNA targeted for cleavage will most often be part of a larger RNAmolecule, often an mRNA molecule. Where relevant, a “part” of an mRNAtarget is a contiguous sequence of an mRNA target of sufficient lengthto allow it to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target ANGPTL3 expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandsequence is selected from the group of sequences provided in any one ofTables 2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14,15A, and 15B and the corresponding nucleotide sequence of the antisensestrand of the sense strand is selected from the group of sequences ofany one of Tables 2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A,13B, 14, 15A, and 15B. In this aspect, one of the two sequences iscomplementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of an ANGPTL3 gene. As such, in this aspect,a dsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand (passenger strand) in any one of Tables2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14, 15A, and15B and the second oligonucleotide is described as the correspondingantisense strand (guide strand) of the sense strand in any one of Tables2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14, 15A, and15B. In one embodiment, the substantially complementary sequences of thedsRNA are contained on separate oligonucleotides. In another embodiment,the substantially complementary sequences of the dsRNA are contained ona single oligonucleotide.

It will be understood that, although the sequences in Tables 2A, 2B, 4A,4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14, 15A, and 15B aredescribed as modified, unmodified, unconjugated. and/or conjugatedsequences, the RNA of the iRNA of the invention e.g., a dsRNA of theinvention, may comprise any one of the sequences set forth in any one ofTables 2A, 2B, 4A, 4B, 5, 7A, 7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14,15A, and 15B that is un-modified, un-conjugated, and/or modified and/orconjugated differently than described therein.

In another aspect, a double-stranded ribonucleic acid (dsRNA) of theinvention for inhibiting expression of ANGPTL3 comprises, consistsessentially of, or consists of a sense strand and an antisense strand,wherein the sense strand comprises the nucleotide sequence5′-usgsucacUfuGfAfAfcucaacucaaL96-3′, and the antisense strand comprisesthe nucleotide sequence 5′-as usUfsgagUfuGfAfguucAfaGfugacasusa-3′; orthe sense strand comprises the nucleotide sequence5′-gsasauauGfuCfAfCfuugaacucaaL96-3′, and the antisense strand comprisesthe nucleotide sequence usUfsgagUfuCfAfagugAfcAfuauucsusu.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., (2001) EMBO J., 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided herein, dsRNAs described herein caninclude at least one strand of a length of minimally 21 nucleotides. Itcan be reasonably expected that shorter duplexes minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences provided herein, and differing in theirability to inhibit the expression of an ANGPTL3gene by not more thanabout 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising thefull sequence, are contemplated to be within the scope of the presentinvention.

In addition, the RNAs described herein identify a site(s) in an ANGPTL3transcript that is susceptible to RISC-mediated cleavage. As such, thepresent invention further features iRNAs that target within thissite(s). As used herein, an iRNA is said to target within a particularsite of an RNA transcript if the iRNA promotes cleavage of thetranscript anywhere within that particular site. Such an iRNA willgenerally include at least about 15 contiguous nucleotides from one ofthe sequences provided herein coupled to additional nucleotide sequencestaken from the region contiguous to the selected sequence in anANGPTL3gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified herein represent effective targetsequences, it is contemplated that further optimization of inhibitionefficiency can be achieved by progressively “walking the window” onenucleotide upstream or downstream of the given sequences to identifysequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified herein,further optimization could be achieved by systematically either addingor removing nucleotides to generate longer or shorter sequences andtesting those sequences generated by walking a window of the longer orshorter size up or down the target RNA from that point. Again, couplingthis approach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art and/or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA agent as described herein can contain one or more mismatches tothe target sequence. In one embodiment, an iRNA as described hereincontains no more than 3 mismatches. If the antisense strand of the iRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch is not located in the center of the region ofcomplementarity. If the antisense strand of the iRNA contains mismatchesto the target sequence, it is preferable that the mismatch be restrictedto be within the last 5 nucleotides from either the 5′- or 3′-end of theregion of complementarity. For example, for a 23 nucleotide iRNA agentthe strand which is complementary to a region of an ANGPTL3 gene,generally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of an ANGPTL3gene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of an ANGPTL3 gene is important, especially if theparticular region of complementarity in an ANGPTL3 gene is known to havepolymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified. iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ONRCH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chico. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA of the invention can also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., (1991) Angewandte Chemie,International Edition, 30:613, and those disclosed by Sanghvi, Y S.,Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

An iRNA of the invention can also be modified to include one or morelocked nucleic acids (LNA). A locked nucleic acid is a nucleotide havinga modified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

An iRNA of the invention can also be modified to include one or morebicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modifiedby the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is anucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments an agent of theinvention may include one or more locked nucleic acids (LNA). A lockednucleic acid is a nucleotide having a modified ribose moiety in whichthe ribose moiety comprises an extra bridge connecting the 2′ and 4′carbons. In other words, an LNA is a nucleotide comprising a bicyclicsugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193). Examples of bicyclic nucleosides for use inthe polynucleotides of the invention include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, the antisense polynucleotide agents ofthe invention include one or more bicyclic nucleosides comprising a 4′to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides,include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

An iRNA of the invention can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and -C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of an iRNA of the invention include a 5′ phosphateor 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimicon the antisense strand of an RNAi agent. Suitable phosphate mimics aredisclosed in, for example US Patent Publication No. 2012/0157511, theentire contents of which are incorporated herein by reference.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., (1989) Proc.Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al.,(1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci.,660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let.,3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. AcidsRes., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanovet al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993)Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al.,(1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides,14:969-973), or adamantane acetic acid (Manoharan et al., (1995)Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al.,(1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J.Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glyciosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Inanother embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-based cleavable linking groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more GalNAc (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disorder of lipid metabolism) can be achieved in anumber of different ways. For example, delivery may be performed bycontacting a cell with an iRNA of the invention either in vitro or invivo. In vivo delivery may also be performed directly by administering acomposition comprising an iRNA, e.g., a dsRNA, to a subject.Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J. et al.,(2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J.et al. (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. etal., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al.(2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A. et al.,(2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem.279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. etal., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamerhas been shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O. et al., (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H. et al., (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al.(2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114),cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328;Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. etal., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNAforms a complex with cyclodextrin for systemic administration. Methodsfor administration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the ANGPTL3 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, e.g.,an inherited disorder, such as such familial hypertriglyceridemia,familial partial lipodystrophy type 1 (FPLD1), or an induced or acquireddisorder, such as a disorder induced or acquired as a result of adisease, disorder or condition (e.g., renal failure), a diet, or intakeof certain drugs (e.g., as a result of highly active antiretroviraltherapy (HAART) used for treating, e.g., AIDS or HIV).

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV) or forsubcutaneous delivery. Another example is compositions that areformulated for direct delivery into the liver, e.g., by infusion intothe liver, such as by continuous pump infusion.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an ANGPTL3 gene. In general,a suitable dose of an iRNA of the invention will be in the range ofabout 0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. Typically, a suitable dose of an iRNA ofthe invention will be in the range of about 0.1 mg/kg to about 5.0mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

A repeat-dose regimine may include administration of a therapeuticamount of iRNA on a regular basis, such as every other day to once ayear. In certain embodiments, the iRNA is administered about once permonth to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as disorders of lipidmetabolism that would benefit from reduction in the expression ofANGPTL3. Such models can be used for in vivo testing of iRNA, as well asfor determining a therapeutically effective dose. Suitable mouse modelsare known in the art and include, for example, an obese (ob/ob) mousecontaining a mutation in the obese (ob) gene (Wiegman et al., (2003)Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of anLDL receptor (LDLR −/− mouse; Ishibashi et al., (1993) J Clin Invest92(2):883-893); diet-induced artherosclerosis mouse model (Ishida etal., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoproteinlipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest.96(6):2555-2568).

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

A. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either aqueous phase, oily phase or itself as aseparate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstras se, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a disorder of lipid metabolism. Examples of such agentsinclude, but are not limited to an anti-inflammatory agent,anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. Inaddition, other substances commonly used to protect the liver, such assilymarin, can also be used in conjunction with the iRNAs describedherein. Other agents useful for treating liver diseases includetelbivudine, entecavir, and protease inhibitors such as telaprevir andother disclosed, for example, in Tung et al., U.S. ApplicationPublication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and inHale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby ANGPTL3 expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VI. Methods of the Invention

The present invention also provides methods of using an iRNA of theinvention and/or a composition containing an iRNA of the invention toreduce and/or inhibit ANGPTL3 expression in a cell. The methods includecontacting the cell with a dsRNA of the invention and maintaining thecell for a time sufficient to obtain degradation of the mRNA transcriptof an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene inthe cell. Reduction in gene expression can be assessed by any methodsknown in the art. For example, a reduction in the expression of ANGPTL3may be determined by determining the mRNA expression level of ANGPTL3using methods routine to one of ordinary skill in the art, e.g.,Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3using methods routine to one of ordinary skill in the art, such asWestern blotting, immunological techniques. A reduction in theexpression of ANGPTL3 may also be assessed indirectly by measuring adecrease in biological activity of ANGPTL3, e.g., a decrease in thelevel of serum lipid, triglycerides, cholesterol and/or free fattyacids.

In the methods of the invention the cell may be contacted in vitro or invivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an ANGPTL3gene. A cell suitable for use in themethods of the invention may be a mammalian cell, e.g., a primate cell(such as a human cell or a non-human primate cell, e.g., a monkey cellor a chimpanzee cell), a non-primate cell (such as a cow cell, a pigcell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbitcell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dogcell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell,or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), ora whale cell. In one embodiment, the cell is a human cell, e.g., a humanliver cell.

ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or about 100%. In preferred embodiments, ANGPTL3 expression isinhibited by at least 20%.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the ANGPTL3 gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection. In certain embodiments, the compositions areadministered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof ANGPTL3, or a therapeutic or prophylactic effect. A depot injectionmay also provide more consistent serum concentrations. Depot injectionsmay include subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an ANGPTL3 gene in a mammal. The methodsinclude administering to the mammal a composition comprising a dsRNAthat targets an ANGPTL3 gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the ANGPTL3 gene, thereby inhibiting expression of theANGPTL3 gene in the cell. Reduction in gene expression can be assessedby any methods known it the art and by methods, e.g. qRT-PCR, describedherein. Reduction in protein production can be assessed by any methodsknown it the art and by methods, e.g. ELISA, described herein. In oneembodiment, a puncture liver biopsy sample serves as the tissue materialfor monitoring the reduction in ANGPTL3 gene and/or protein expression.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of ANGPTL3expression, in a therapeutically effective amount of an iRNA targetingan ANGPTL3 gene or a pharmaceutical composition comprising an iRNAtargeting an ANGPTL3 gene.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition ofANGPTL3 gene expression are those having a disorder of lipid metabolism,e.g., an inherited disorder of lipid metabolism or an acquired disorderof lipid metabolism. In one embodiment, a subject having disorder oflipid metabolism has hyperlipidemia. In another embodiment, a subjecthaving a disorder of lipid metabolism has hypertriglyceridemia.Treatment of a subject that would benefit from a reduction and/orinhibition of ANGPTL3 gene expression includes therapeutic treatment(e.g., a subject is having eruptive xanthomas) and prophylactictreatment (e.g., the subject is not having eruptive xanthomas or asubject may be at risk of developing eruptive xanthomas).

The invention further provides methods for the use of an iRNA or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of ANGPTL3 expression,e.g., a subject having a disorder of lipid metabolism, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating thesedisorders. For example, in certain embodiments, an iRNA targetingANGPTL3 is administered in combination with, e.g., an agent useful intreating a disorder of lipid metabolism as described elsewhere herein.For example, additional agents suitable for treating a subject thatwould benefit from reduction in ANGPTL3 expression, e.g., a subjecthaving a disorder of lipid metabolism, may include agents that lower oneor more serum lipids. Non-limiting examples of such agents may includecholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors,e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin(Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev),pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor),and simvastatin (Zocor). Other agents useful in treating a disorder oflipid metabolism may include bile sequestering agents, such ascholestyramine and other resins; VLDL secretion inhibitors, such asniacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterolacyl transferase inhibitors; farnesoid X receptor antagonists; sterolregulatory binding protein cleavage activating protein (SCAP)activators; microsomal triglyceride transfer protein (MTP) inhibitors;ApoE-related peptide; and therapeutic antibodies against ANGPTL3. Theadditional therapeutic agents may also include agents that raise highdensity lipoprotein (HDL), such as cholesteryl ester transfer protein(CETP) inhibitors. Furthermore, the additional therapeutic agents mayalso include dietary supplements, e.g., fish oil. The iRNA andadditional therapeutic agents may be administered at the same timeand/or in the same combination, e.g., parenterally, or the additionaltherapeutic agent can be administered as part of a separate compositionor at separate times and/or by another method known in the art ordescribed herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target ANGPTL3 gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24hours, 28, 32, or about 36 hours. In one embodiment, expression of thetarget ANGPTL3 gene is decreased for an extended duration, e.g., atleast about two, three, four days or more, e.g., about one week, twoweeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the targetANGPTL3gene. Compositions and methods for inhibiting the expression ofthese genes using iRNAs can be prepared and performed as describedherein.

Administration of the dsRNA according to the methods of the inventionmay result in a reduction of the severity, signs, symptoms, and/ormarkers of such diseases or disorders in a patient with a disorder oflipid metabolism. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of a disorder of lipid metabolism may beassessed, for example, by periodic monitoring of one or more serum lipidlevels. Comparisons of the later readings with the initial readingsprovide a physician an indication of whether the treatment is effective.It is well within the ability of one skilled in the art to monitorefficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. In connection with theadministration of an iRNA targeting ANGPTL3 or pharmaceuticalcomposition thereof, “effective against” a disorder of lipid metabolismindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as a improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating disorder of lipid metabolisms and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). Any positive change resulting in e.g., lessening of severity ofdisease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg to about 50 mg/kg. Typically, a suitable dose of aniRNA of the invention will be in the range of about 0.1 mg/kg to about5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

The iRNA can be administered by intravenous infusion over a period oftime, on a regular basis. In certain embodiments, after an initialtreatment regimen, the treatments can be administered on a less frequentbasis. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in acell, tissue, blood, urine or other compartment of the patient by atleast about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or at least about 99% or more. In a preferredembodiment, administration of the iRNA can reduce ANGPTL3 levels, e.g.,in a cell, tissue, blood, urine or other compartment of the patient byat least 20%.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired daily dose of iRNA to a subject. The injections may berepeated over a period of time. The administration may be repeated on aregular basis. In certain embodiments, after an initial treatmentregimen, the treatments can be administered on a less frequent basis. Arepeat-dose regimine may include administration of a therapeutic amountof iRNA on a regular basis, such as every other day or to once a year.In certain embodiments, the iRNA is administered about once per month toabout once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. iRNA Design, Synthesis, Selection, and In VitroEvaluation

This Example describes methods for the design, synthesis, selection, andin vitro evaluation of ANGPTL3 iRNA agents (also see, PCT publication,WO 2012/177784, the entire contents of which is incorporated herein byreference).

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

A set of siRNAs targeting the human ANGPTL3, “angiopoietin-like 3”(human: NCBI refseqID NM_014995; NCBI GeneID: 27329), as well astoxicology-species ANGPTL3 orthologs (cynomolgus monkey: XM_005543185;mouse: NM_013913; rat, NM_001025065) were designed using custom R andPython scripts. The human ANGPTL3 REFSEQ mRNA has a length of 2951bases. The rationale and method for the set of siRNA designs is asfollows: the predicted efficacy for every potential 19mer siRNA fromposition 81 through position 2951 (the coding region and 3′ UTR) wasdetermined with a linear model derived the direct measure of mRNAknockdown from more than 20,000 distinct siRNA designs targeting a largenumber of vertebrate genes. Subsets of the ANGPTL3 siRNAs were designedwith perfect or near-perfect matches between human, cynomolgus andrhesus monkey. A further subset was designed with perfect ornear-perfect matches to mouse and rat ANGPTL3 orthologs. For each strandof the siRNA, a custom Python script was used in a brute force search tomeasure the number and positions of mismatches between the siRNA and allpotential alignments in the target species transcriptome. Extra weightwas given to mismatches in the seed region, defined here as positions2-9 of the antisense oligonucleotide, as well the cleavage site of thesiRNA, defined here as positions 10-11 of the antisense oligonucleotide.The relative weight of the mismatches was 2.8; 1.2:1 for seedmismatches, cleavage site, and other positions up through antisenseposition 19. Mismatches in the first position were ignored. Aspecificity score was calculated for each strand by summing the value ofeach weighted mismatch. Preference was given to siRNAs whose antisensescore in human and cynomolgus monkey was >=3.0 and predicted efficacywas >=70% knockdown of the ANPTL3 transcript.

Synthesis of ANGPTL3 Sequences

Synthesis of ANGPTL3 Single Strands and Duplexes

ANGPTL3 siRNA sequences were synthesized at 1 umol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500° A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modifiednucleosides were introduced in the sequences using the correspondingphosphoramidites. Synthesis of 3′ GalNAc conjugated single strands wasperformed on a GalNAc modified CPG support. Custom CPG universal solidsupport was used for the synthesis of antisense single strands. Couplingtime for all phosphoramidites (100 mM in acetonitrile) was 5 minemploying 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M inacetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass.,USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3minutes. All sequences were synthesized with final removal of the DMTgroup (“DMT off”).

Upon completion of the solid phase synthesis, single strands werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagent at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 uL ofdimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent was added and thesolution was incubated for additional 20 min at 60° C. At the end ofcleavage and deprotection step, the synthesis plate was allowed to cometo room temperature and was precipitated by addition of 1 mL ofacetontile:ethanol mixture (9:1). The plates were cooled at −80° C. for2 hrs and the supernatant decanted carefully with the aid of amulti-channel pipette. The oligonucleotide pellet was re-suspended in 20mM NaOAc buffer and were desalted using a 5 mL HiTrap size exclusioncolumn (GE Healthcare) on an AKTA Purifier System equipped with an A905autosampler and a Frac 950 fraction collector. Desalted samples werecollected in 96 well plates. Samples from each sequence were analyzed byLC-MS to confirm the identity, UV (260 nm) for quantification and aselected set of samples by IEX chromatography to determine purity.

Annealing of ANGPTL3 single strands was performed on a Tecan liquidhandling robot. Equimolar mixture of sense and antisense single strandswere combined and annealed in 96 well plates. After combining thecomplementary single strands, the 96 well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10 uM in 1×PBS and then submitted for in vitroscreening assays.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]- 4-hydroxyprolinolHyp-(GalNAc-alkyl)3 dT 2{grave over ( )}-deoxythymidine-3{grave over( )}-phosphate dC 2{grave over ( )}-deoxycytidine-3{grave over( )}-phosphate Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Tgn) Thymidine-glycol nucleic acid (GNA)S-Isomer P Phosphate VP Vinyl-phosphate (Aam) 2{grave over( )}-O-(N-methylacetamide)adenosine-3{grave over ( )}-phosphate (Aams)2{grave over ( )}-O-(N-methylacetamide)adenosine-3{grave over( )}-phosphorothioate (Cgn) Cytidine-glycol nucleic acid (GNA)

Example 2. Lead Selection and Evaluation

Based on the results of various in vitro and in vivo analyses (see, PCTpublication, WO 2012/177784, the entire contents of which areincorporated herein by reference), the parent sequences of AD-52981(sense sequence: ACAUAUUUGAUCAGUCUUUUU (SEQ ID NO: 20); antisensesequence: AAAAAGACUGAUCAAAUAUGUUG) (SEQ ID NO: 21) were selected formodification and further evaluation in vivo.

Therefore, using the parent sequences of AD-52981, AD-57927 (sensesequence: AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 70);antisense sequence: asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg) (SEQ ID NO:71) was synthesized as described above.

The effect of AD-57927 in vivo was evaluated by subcutaneouslyadministering C57BL/6 female mice with a single 30, 10, or 3 mg/kg doseof AD-52981 (sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQID NO: 72); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg)(SEQ ID NO: 73), AD-57927, or PBS control. At 72 hours post dose,animals were sacrificed and liver ANGPTL3 mRNA levels were determined.Surprisingly, and as shown in FIG. 1, AD-57927 (“ANG-GalNAc Optimized”)decreased ANGPTL3 mRNA levels by about 10-fold more than the parent iRNAagent, AD-52981 (“ANG-GalNAc”).

The ability of AD-57927 to suppress expression of ANGPTL3 protein and todecrease the levels of tryglycerides, LDL cholesterol, and totalcholesterol in vivo was also assessed using a multi-dose regimen. ob/obfemale mice were subcutaneously administered AD-57927 at 3 mg/kg everyday for 5 days on week 1 followed by a dose of 3 mg/kg two times perweek on weeks 2-4 (qd×5; qw×6). Animals were bled on days 0, 4, 8, 11,15, 18, 22, and 25, and sacrificed on day 29. ANPTL3 protein levels weremeasured by ELISA. Triglycerides, LDL cholesterol, and total cholesterolwere measured using an Olympus serum analyzer. As shown in FIG. 2, themaximal knockdown of ANGPTL3 protein by AD-57927 was 99%. The maximallevels of triglyceride lowering by AD-57927 was 98% (FIG. 3), themaximal LDL lowering by AD-57927 was 88% (FIG. 4), and the maximal totalcholesterol lowering by AD-57927 was 64% (FIG. 5). All of the datapresented in FIGS. 2-5 is relative to predose levels.

In summary, AD-57927 was demonstrated to reduce ANGPTL3 mRNA levelsabout 10-fold more that the parent sequence. AD-57927 was alsodemonstrated to reduce ANGPTL3 in a dose responsive manner (see FIG. 1)and the efficacy of AD-57927 was further improved upon multi-doseadministration resulting in greater than 95% reduction of serum ANPTL3protein in the ob/ob mouse model of mixed hyperlipidemia. Furthermore,multi-dose administration of AD-57927 at 3 mg/kg eliminated circulatingserum ANGPLT3 protein as measured by ANGPTL3 specific ELISA assay, whichresulted in a >95% reduction in TGs, a >85% reduction in LDLcholesterol, and a >60% reduction in total cholesterol in the ob/obmouse model of hyperlipidemia.

Example 3. iRNA Design, Synthesis, Selection, and In Vitro Evaluation

This Example describes methods for the design, synthesis, selection, andin vitro evaluation of additional ANGPTL3 iRNA agents.

In Vitro Screening:

Cell Culture and Transfections:

Hep3b cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium(Gibco) supplemented with 10% FBS (ATCC) before being released from theplate by trypsinization. Transfection was carried out by adding 4.9 μlof Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of each siRNA duplex to anindividual well in a 384-well plate. The mixture was then incubated atroom temperature for 20 minutes. 40 μl of complete growth mediacontaining 5,000 Hep3b cells were then added to the siRNA mixture. Cellswere incubated for 24 hrs prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were lysed in 75 μl of Lysis/Binding Buffer containing 3 μL ofbeads per well and mixed for 10 minutes on an electrostatic shaker. Thewashing steps were automated on a Biotek EL406, using a magnetic platesupport. Beads were washed (90 μL) once in Buffer A, once in Buffer B,and twice in Buffer E, with aspiration steps in between. Following afinal aspiration, complete 10 μL RT mixture was added to each well, asdescribed below.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl, 10× Buffer, 0.4 μl 25× dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2Oper reaction were added per well. Plates were sealed, agitated for 10minutes on an electrostatic shaker, and then incubated at 37 degrees C.for 2 hours. Following this, the plates were agitated at 80 degrees C.for 8 minutes.

Real time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl of human GAPDHTaqMan Probe (4326317E), 0.5 μl human AngPTL3 (Hs00205581_m1), 2 μlnuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well plates (Roche cat #04887301001).Real time PCR was done in a LightCycler480 Real Time PCR system (Roche)using the ΔΔCt(RQ) assay. Each duplex was tested in at least twoindependent transfections, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM nonspecific siRNA, or mock transfected cells.

A series of ANGPLT3 iRNAs containing various chemical modificationsbased on the sense sequence 5′-ACAUAUUUGAUCAGUCUUUUU-3′ (SEQ ID NO: 20)and the antisense sequence 5′-AAAAAGACUGAUCAAAUAUGUUG-3′ (SEQ ID NO: 21)were tested. The chemical modifications of the sequences are shown inTables 2A and 2B. The results from the assays are shown in Table 3.

Additional iRNA agents targeting the ANGPTL3 gene were synthesized asdescribed above. A detailed list of the additional modified ANGPTL3sense and antisense strand sequences is shown in Tables 4A, 4B, 7A, and7B and a detailed list of the unmodified ANGPTL3 sense and antisensestrand sequences is shown in Tables 5 and 7C.

TABLE 2A ANGPTL3 Modified Sequences SEQ SEQ ID ID Duplex NameSense Sequence 5′ to 3′ NO Antisense Sequence 5′ to 3′ NO AD-57927AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  74asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 170 AD-63131ascsauauuugaUfCfagucuuuuuL96  75 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg171 AD-63132 ascsauauuugadTdCagucuuuuuL96  76asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 172 AD-63133AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  77asAfsaAfagaCfuGfaucAfaAfuAfugususg 173 AD-63134ascsauaUfuuGfaUfCfagUfCfuuuuuL96  78 asAfsaaaGfacUfgaucAfaAfuaugususg174 AD-63136 Y44AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  79asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 175 AD-63137ascsauauUfuGfAfUfcaguCfuudTuuL96  80asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 176 AD-63138ascsauauuugadTcagucuuuuuL96  81 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 177AD-63139 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  82asAfsaAfagaCfugaucAfaAfuAfugususg 178 AD-63140ascsauaUfuugaUfcagucuuuuuL96  83 asAfsaaaGfacUfgaucAfaAfuaugususg 179AD-63142 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  84PasAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 180 AD-63143ascsauauUfuGfAfUfcaguCfdTuuuuL96  85asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 181 AD-63144ascsauauuugadTcaguc(Tgn)uuuuL96  86 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg182 AD-63145 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  87asAfsaAfagacuGfaucAfaAfuAfugususg 183 AD-63146ascsauaUfuuGfaUfCfagUfCfuudTuuL96  88 asAfsaaaGfacUfgaucAfaAfuaugususg184 AD-63148 Y44AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  89PasAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 185 AD-63149ascsauauUfuGfAfdTcaguCfuuuuuL96  90 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg186 AD-63150 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  91asAfsaAfaGfacuGfaucAfaAfuAfuGfususg 187 AD-63151AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  92asAfsaaaGfacUfgaucAfaAfuaugususg 188 AD-63152ascsauaUfuuGfaUfCfagUfCfdTuuuuL96  93 asAfsaaaGfacUfgaucAfaAfuaugususg189 AD-63153 asusAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  94PasAfsaAfaGfaCfuGfaucAfaAfuAfusgsu 190 AD-63154ascsauaUfuugAfUfcAfguCfuuuuuL96  95 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg191 AD-63155 ascsauaUfuugaUfcagucuudTuuL96  96asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 192 AD-63156AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  97asAfsaaaGfaCfuGfaucAfaAfuauGfususg 193 AD-63157AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96  98asAfsaaaGfacugaucAfaAfuaugususg 194 AD-63158ascsauaUfuuGfadTCfagUfCfuuuuuL96  99 asAfsaaaGfacUfgaucAfaAfuaugususg195 AD-63160 ascsauauUfuGfAfUfcaguCfuuuuuL96 100asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 196 AD-63161ascsauaUfuugaUfcagucdTuuuuL96 101 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg197 AD-63162 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 102asAfsaAfaGfaCfuGfaucAfaAfuaugususg 198 AD-63163AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 103asAfsaaaGfaCfUfgaucAfaAfuaugususg 199 AD-63164ascsauaUfuugaUfcagucuudTuuL96 104 asAfsaaaGfacUfgaucAfaAfuaugususg 200AD-63165 ascsauaUfuuGfaUfCfagUfCfuuuuuL96 105asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 201 AD-63166ascsauaUfuugaUfdCagucuuuuuL96 106 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg202 AD-63167 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 107asAfsaaaGfacuGfaucAfaAfuauGfususg 203 AD-63168ascsauaUfuugAfUfcAfguCfuuuuuL96 108 asAfsaaaGfacUfgaucAfaAfuaugususg 204AD-63169 ascsauaUfuugaUfcagucdTuuuuL96 109asAfsaaaGfacUfgaucAfaAfuaugususg 205 AD-63170ascsAfuAfuuuGfAfUfcAfguCfUfuuuuL96 110asAfsAfaaGfaCfuGfaucAfAfAfuAfuGfususg 206 AD-63171ascsauaUfuugaUfcagucuuuuuL96 111 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg207 AD-63172 ascsauauuugaUfdCagucuuuuuL96 112asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 208 AD-63173AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 113asAfsaaaGfaCfuGfaucAfaAfuaugususg 209 AD-63174ascsauauUfuGfAfUfcaguCfuuuuuL96 114 asAfsaaaGfacUfgaucAfaAfuaugususg 210AD-63175 ascsauaUfuugaUfdCagucuuuuuL96 115asAfsaaaGfacUfgaucAfaAfuaugususg 211 AD-63176ascsAfuAfuuuGfAfUfcAfguCfUfuuuuL96 116asAfsAfAfaGfaCfuGfaucAfAfAfuAfuGfususg 212 AD-63177AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 117asAfsa(Aam)aGfaCfuGfaucAfaAfuAfuGfususg 213 AD-63179ascsAfuAfuuuGfAfUfcAfguCfUfuuuuL96 118asAfsAfaaGfaCfuGfaucAfAfAfuAfugususg 214 AD-63180AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 119as(Aams)aAfaGfaCfuGfaucAfaAfu(Aam)uGfususg 215 AD-63181ascsAfuAfuuuGfAfUfcAfguCfUfuuuuL96 120asAfsAfAfaGfaCfuGfaucAfAfAfuAfugususg 216 AD-63182AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 121asAfsa(Aam)aGfaCfuGfaucAfaAfu(Aam)uGfususg 217 AD-63183AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 122asasaAfaGfaCfuGfaucAfaAfuAfuGfususg 218 AD-63185AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 123asAfsaAfagaCfuGfaucAfaAfuAfuGfususg 219 AD-63186AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 124asAfsaAfaGfaCfuGfaucaaAfuAfuGfususg 220 AD-63187AfscsAfuAfuUfuGfAfucAfgUfcUfuUfuUfL96 125asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 221 AD-63188AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 126as(Aams)aAfaGfaCfuGfaucAfaAfuAfuGfususg 222 AD-64744ascsauauuugadTcagucdTuuuuL96 127 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg223 AD-64745 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 128asAfsaaaGfacuGfaucAfaauaugususg 224 AD-64746AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 129asdAsaaagacugaucdAadAuaugususg 225 AD-64747ascsauauuuGfaUfcagUfcuuUfuuL96 130 asAfsaaaGfacuGfaucAfaAfuaugususg 226AD-64748 ascsauauuugadTcagucdAuuuuL96 131 asdAsaaagacudGaucdAaauaugususg227 AD-64749 ascsauauuuGfaUfcagUfcuuUfuuL96 132asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 228 AD-64750ascsauauuugadTdCagucdTuuuuL96 133 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg229 AD-64751 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 134asdAsaaadGacudGaucdAaauaugususg 230 AD-64752ascsauauuuGfaUfcagUfcuuuuuL96 135 asAfsaaaGfacuGfaucAfaAfuaugususg 231AD-64753 ascsauauuugadTcagucdAuuuuL96 136 asdAsaaadGacugaucdAaauaugususg232 AD-64754 ascsauauuuGfaUfcagUfcuuuuuL96 137asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 233 AD-64755ascsauauuugadTdCagucdCuuuuL96 138 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg234 AD-64756 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 139asAfsaaagacugaucAfaauaugususg 235 AD-64757AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 140 asAsaaagacudGaucdAaauaugususg236 AD-64758 ascsauauuuGfaUfcagUfcuuUfuuL96 141asAfsaaaGfacUfGfaucAfaAfuaugususg 237 AD-64759ascsauauuugadTcagucdAuuuuL96 142 asdAsaaadGacudGaucdAaauaugususg 238AD-64760 ascsauauuuuadTcagucuuuuuL96 143asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 239 AD-64761ascsauauuugadTcagucdGuuuuL96 144 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg240 AD-64762 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 145asdAsaaagacugaucdAaauaugususg 241 AD-64763AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 146 asdAsaaagacudGaucAaauaugususg242 AD-64764 ascsauauuuGfaUfcagUfcuuuuuL96 147asAfsaaaGfacUfGfaucAfaAfuaugususg 243 AD-64765ascsauauuugaUcaguc(Tgn)uuuuL96 148 asdAsaaagacudGaucdAaauaugususg 244AD-64766 ascsauauuugadTcagu(Cgn)uuuuuL96 149asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 245 AD-64767ascsauauuugadTcagucdCuuuuL96 150 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg246 AD-64768 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 151asasaaagacudGaucdAaauaugususg 247 AD-64769AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 152 asAsaaagacudGaucAaauaugususg248 AD-64770 ascsauauuugadTcaguc(Tgn)uuuuL96 153asdAsaaagacudGaucdAaauaugususg 249 AD-64771ascsauauuugaUcaguc(Tgn)uuuuL96 154 asdAsaaadGacugaucdAaauaugususg 250AD-64772 ascsauauuugadTcagucdAuuuuL96 155asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 251 AD-64773AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 156 asasaaadGacugaucdAaauaugususg252 AD-64774 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 157asAsaaadGacugaucAaauaugususg 253 AD-64775ascsauauuugadTcaguc(Tgn)uuuuL96 158 asdAsaaadGacugaucdAaauaugususg 254AD-64776 ascsauauuugaUcaguc(Tgn)uuuuL96 159asdAsaaadGacudGaucdAaauaugususg 255 AD-64777ascsauauuugadTcagucu(Tgn)uuuL96 160 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg256 AD-64778 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 161asAfsaaaGfacuGfaucAfaAfuaugususg 257 AD-64779AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 162asdAsaaagacudGaucdAaauaugususg 258 AD-64780AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 163PasdAsaaagacudGaucdAaauaugususg 259 AD-64781ascsauauuugadTcaguc(Tgn)uuuuL96 164 asdAsaaadGacudGaucdAaauaugususg 260AD-64782 ascsauauuugaUcaguc(Tgn)uuuuL96 165asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 261 AD-64783AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 166asAfsaaaGfacUfGfaucAfaAfuaugususg 262 AD-64784AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 167asdAsaaadGacugaucdAaauaugususg 263 AD-64785AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 168PasasaaagacudGaucdAaauaugususg 264 AD-64786ascsauauuugadTcaguc(Tgn)uuuuL96 169 PasdAsaaagacudGaucdAaauaugususg 265

TABLE 2B ANGPTL3 Modified Sequences SEQ SEQ ID ID Duplex NameSense Sequence 5′ to 3′ NO Antisense Sequence 5′ to 3′ NO AD-57927AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  74asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 170 AD-63131ascsauauuugaUfCfagucuuuuu  75 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 171AD-63132 ascsauauuugadTdCagucuuuuu  76asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 172 AD-63133AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  77asAfsaAfagaCfuGfaucAfaAfuAfugususg 173 AD-63134ascsauaUfuuGfaUfCfagUfCfuuuuu  78 asAfsaaaGfacUfgaucAfaAfuaugususg 174AD-63136 Y44AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  79asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 175 AD-63137ascsauauUfuGfAfUfcaguCfuudTuu  80 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg176 AD-63138 ascsauauuugadTcagucuuuuu  81asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 177 AD-63139AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  82asAfsaAfagaCfugaucAfaAfuAfugususg 178 AD-63140 ascsauaUfuugaUfcagucuuuuu 83 asAfsaaaGfacUfgaucAfaAfuaugususg 179 AD-63142AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  84PasAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 180 AD-63143ascsauauUfuGfAfUfcaguCfdTuuuu  85 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg181 AD-63144 ascsauauuugadTcaguc(Tgn)uuuu  86asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 182 AD-63145AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  87asAfsaAfagacuGfaucAfaAfuAfugususg 183 AD-63146ascsauaUfuuGfaUfCfagUfCfuudTuu  88 asAfsaaaGfacUfgaucAfaAfuaugususg 184AD-63148 Y44AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  89PasAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 185 AD-63149ascsauauUfuGfAfdTcaguCfuuuuu  90 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg186 AD-63150 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  91asAfsaAfaGfacuGfaucAfaAfuAfuGfususg 187 AD-63151AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  92 asAfsaaaGfacUfgaucAfaAfuaugususg188 AD-63152 ascsauaUfuuGfaUfCfagUfCfdTuuuu  93asAfsaaaGfacUfgaucAfaAfuaugususg 189 AD-63153asusAfuUfuGfAfUfcAfgUfcUfuUfuUf  94 PasAfsaAfaGfaCfuGfaucAfaAfuAfusgsu190 AD-63154 ascsauaUfuugAfUfcAfguCfuuuuu  95asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 191 AD-63155ascsauaUfuugaUfcagucuudTuu  96 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 192AD-63156 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  97asAfsaaaGfaCfuGfaucAfaAfuauGfususg 193 AD-63157AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf  98 asAfsaaaGfacugaucAfaAfuaugususg194 AD-63158 ascsauaUfuuGfadTCfagUfCfuuuuu  99asAfsaaaGfacUfgaucAfaAfuaugususg 195 AD-63160ascsauauUfuGfAfUfcaguCfuuuuu 100 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg196 AD-63161 ascsauaUfuugaUfcagucdTuuuu 101asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 197 AD-63162AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 102asAfsaAfaGfaCfuGfaucAfaAfuaugususg 198 AD-63163AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 103asAfsaaaGfaCfUfgaucAfaAfuaugususg 199 AD-63164ascsauaUfuugaUfcagucuudTuu 104 asAfsaaaGfacUfgaucAfaAfuaugususg 200AD-63165 ascsauaUfuuGfaUfCfagUfCfuuuuu 105asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 201 AD-63166ascsauaUfuugaUfdCagucuuuuu 106 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 202AD-63167 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 107asAfsaaaGfacuGfaucAfaAfuauGfususg 203 AD-63168ascsauaUfuugAfUfcAfguCfuuuuu 108 asAfsaaaGfacUfgaucAfaAfuaugususg 204AD-63169 ascsauaUfuugaUfcagucdTuuuu 109 asAfsaaaGfacUfgaucAfaAfuaugususg205 AD-63170 ascsAfuAfuuuGfAfUfcAfguCfUfuuuu 110asAfsAfaaGfaCfuGfaucAfAfAfuAfuGfususg 206 AD-63171ascsauaUfuugaUfcagucuuuuu 111 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 207AD-63172 ascsauauuugaUfdCagucuuuuu 112asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 208 AD-63173AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 113asAfsaaaGfaCfuGfaucAfaAfuaugususg 209 AD-63174ascsauauUfuGfAfUfcaguCfuuuuu 114 asAfsaaaGfacUfgaucAfaAfuaugususg 210AD-63175 ascsauaUfuugaUfdCagucuuuuu 115 asAfsaaaGfacUfgaucAfaAfuaugususg211 AD-63176 ascsAfuAfuuuGfAfUfcAfguCfUfuuuu 116asAfsAfAfaGfaCfuGfaucAfAfAfuAfuGfususg 212 AD-63177AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 117asAfsa(Aam)aGfaCfuGfaucAfaAfuAfuGfususg 213 AD-63179ascsAfuAfuuuGfAfUfcAfguCfUfuuuu 118 asAfsAfaaGfaCfuGfaucAfAfAfuAfugususg214 AD-63180 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 119as(Aams)aAfaGfaCfuGfaucAfaAfu(Aam)uGfususg 215 AD-63181ascsAfuAfuuuGfAfUfcAfguCfUfuuuu 120asAfsAfAfaGfaCfuGfaucAfAfAfuAfugususg 216 AD-63182AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 121asAfsa(Aam)aGfaCfuGfaucAfaAfu(Aam)uGfususg 217 AD-63183AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 122asasaAfaGfaCfuGfaucAfaAfuAfuGfususg 218 AD-63185AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 123asAfsaAfagaCfuGfaucAfaAfuAfuGfususg 219 AD-63186AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 124asAfsaAfaGfaCfuGfaucaaAfuAfuGfususg 220 AD-63187AfscsAfuAfuUfuGfAfucAfgUfcUfuUfuUf 125asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 221 AD-63188AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 126as(Aams)aAfaGfaCfuGfaucAfaAfuAfuGfususg 222 AD-64744ascsauauuugadTcagucdTuuuu 127 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 223AD-64745 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 128asAfsaaaGfacuGfaucAfaauaugususg 224 AD-64746AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 129 asdAsaaagacugaucdAadAuaugususg225 AD-64747 ascsauauuuGfaUfcagUfcuuUfuu 130asAfsaaaGfacuGfaucAfaAfuaugususg 226 AD-64748 ascsauauuugadTcagucdAuuuu131 asdAsaaagacudGaucdAaauaugususg 227 AD-64749ascsauauuuGfaUfcagUfcuuUfuu 132 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 228AD-64750 ascsauauuugadTdCagucdTuuuu 133asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 229 AD-64751AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 134 asdAsaaadGacudGaucdAaauaugususg230 AD-64752 ascsauauuuGfaUfcagUfcuuuuu 135asAfsaaaGfacuGfaucAfaAfuaugususg 231 AD-64753 ascsauauuugadTcagucdAuuuu136 asdAsaaadGacugaucdAaauaugususg 232 AD-64754ascsauauuuGfaUfcagUfcuuuuu 137 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 233AD-64755 ascsauauuugadTdCagucdCuuuu 138asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 234 AD-64756AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 139 asAfsaaagacugaucAfaauaugususg235 AD-64757 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 140asAsaaagacudGaucdAaauaugususg 236 AD-64758 ascsauauuuGfaUfcagUfcuuUfuu141 asAfsaaaGfacUfGfaucAfaAfuaugususg 237 AD-64759ascsauauuugadTcagucdAuuuu 142 asdAsaaadGacudGaucdAaauaugususg 238AD-64760 ascsauauuuuadTcagucuuuuu 143asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 239 AD-64761ascsauauuugadTcagucdGuuuu 144 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 240AD-64762 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 145asdAsaaagacugaucdAaauaugususg 241 AD-64763AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 146 asdAsaaagacudGaucAaauaugususg242 AD-64764 ascsauauuuGfaUfcagUfcuuuuu 147asAfsaaaGfacUfGfaucAfaAfuaugususg 243 AD-64765ascsauauuugaUcaguc(Tgn)uuuu 148 asdAsaaagacudGaucdAaauaugususg 244AD-64766 ascsauauuugadTcagu(Cgn)uuuuu 149asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 245 AD-64767ascsauauuugadTcagucdCuuuu 150 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 246AD-64768 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 151asasaaagacudGaucdAaauaugususg 247 AD-64769AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 152 asAsaaagacudGaucAaauaugususg 248AD-64770 ascsauauuugadTcaguc(Tgn)uuuu 153 asdAsaaagacudGaucdAaauaugususg249 AD-64771 ascsauauuugaUcaguc(Tgn)uuuu 154asdAsaaadGacugaucdAaauaugususg 250 AD-64772 ascsauauuugadTcagucdAuuuu155 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 251 AD-64773AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 156 asasaaadGacugaucdAaauaugususg252 AD-64774 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 157asAsaaadGacugaucAaauaugususg 253 AD-64775 ascsauauuugadTcaguc(Tgn)uuuu158 asdAsaaadGacugaucdAaauaugususg 254 AD-64776ascsauauuugaUcaguc(Tgn)uuuu 159 asdAsaaadGacudGaucdAaauaugususg 255AD-64777 ascsauauuugadTcagucu(Tgn)uuu 160asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 256 AD-64778AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 161 asAfsaaaGfacuGfaucAfaAfuaugususg257 AD-64779 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 162asdAsaaagacudGaucdAaauaugususg 258 AD-64780AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 163 PasdAsaaagacudGaucdAaauaugususg259 AD-64781 ascsauauuugadTcaguc(Tgn)uuuu 164asdAsaaadGacudGaucdAaauaugususg 260 AD-64782 ascsauauuugaUcaguc(Tgn)uuuu165 asAfsaAfaGfaCfuGfaucAfaAfuAfuGfususg 261 AD-64783AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 166asAfsaaaGfacUfGfaucAfaAfuaugususg 262 AD-64784AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 167 asdAsaaadGacugaucdAaauaugususg263 AD-64785 AfscsAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf 168PasasaaagacudGaucdAaauaugususg 264 AD-64786 ascsauauuugadTcaguc(Tgn)uuuu169 PasdAsaaagacudGaucdAaauaugususg 265

TABLE 3 AngPTL3 Single Dose Screen in Hep3B cells. Data are expressed aspercent message remaining relative to AD-1955 non-targeting control. 10nM 0.1 nM 10 nM 0.1 nM Duple Name AVG AVG STDEV STDEV AD-57927 3.9 4.31.9 1.0 AD-63131 14.3 15.6 7.7 6.0 AD-63132 12.5 10.6 2.3 3.0 AD-631338.6 7.5 1.4 1.9 AD-63134 17.3 15.9 2.8 1.9 AD-63136 2.7 4.3 0.9 1.4AD-63137 2.9 3.4 0.9 0.7 AD-63138 13.6 18.8 3.9 3.9 AD-63139 7.5 9.0 2.61.7 AD-63140 13.0 15.0 8.6 6.7 AD-63142 3.2 3.9 0.7 0.4 AD-63143 5.3 4.62.3 2.2 AD-63144 5.6 6.6 2.9 3.0 AD-63145 7.3 6.6 1.8 0.9 AD-63146 17.514.5 2.5 4.6 AD-63148 2.9 3.1 1.4 2.2 AD-63149 6.3 7.2 2.1 1.4 AD-631504.8 6.5 1.9 2.5 AD-63151 4.7 4.7 2.3 2.9 AD-63152 10.3 12.2 2.0 1.5AD-63153 5.1 4.9 1.9 2.4 AD-63154 9.4 7.9 3.7 3.0 AD-63155 13.4 13.2 5.14.3 AD-63156 3.3 4.4 1.1 1.4 AD-63157 2.6 4.4 2.1 1.8 AD-63158 16.7 23.98.7 7.2 AD-63160 3.5 3.7 1.9 1.2 AD-63161 11.7 12.6 5.7 1.2 AD-63162 3.96.2 1.2 1.5 AD-63163 3.7 3.6 2.5 1.7 AD-63164 11.3 13.6 8.2 5.0 AD-6316512.5 13.3 3.7 5.2 AD-63166 8.8 13.6 5.2 9.4 AD-63167 3.3 2.8 1.2 0.9AD-63168 7.3 9.0 6.0 5.0 AD-63169 10.8 12.1 8.7 7.2 AD-63170 3.7 3.8 1.10.9 AD-63171 12.5 16.4 6.9 2.6 AD-63172 8.7 10.1 7.2 4.7 AD-63173 3.03.6 1.3 2.5 AD-63174 2.8 3.7 1.5 1.6 AD-63175 5.2 3.4 2.8 2.6 AD-631763.3 4.2 0.9 0.4 AD-63177 5.3 7.3 2.3 0.6 AD-63179 3.0 4.0 0.9 0.9AD-63180 66.8 92.2 22.2 13.5 AD-63181 4.8 5.4 1.9 1.0 AD-63182 14.7 23.14.7 6.4 AD-63183 11.8 14.3 5.5 4.7 AD-63185 7.9 8.8 0.7 2.8 AD-6318657.6 72.0 12.1 15.7 AD-63187 9.8 14.1 4.8 2.8 AD-63188 30.9 49.8 20.78.3 AD-64744 16.6 57.4 1.4 9.8 AD-64745 12.0 58.3 2.2 17.5 AD-64746 67.399.4 6.1 14.5 AD-64747 18.0 65.1 2.5 2.2 AD-64748 69.0 87.2 5.2 5.6AD-64749 16.4 70.5 5.0 11.2 AD-64750 12.3 44.3 4.6 7.0 AD-64751 57.987.5 7.5 7.4 AD-64752 18.7 54.1 4.0 16.1 AD-64753 57.6 81.9 5.1 4.4AD-64754 21.2 72.3 2.4 6.7 AD-64755 9.0 32.7 2.3 9.1 AD-64756 17.2 53.73.9 7.9 AD-64757 89.0 92.0 4.4 5.8 AD-64758 23.5 66.1 10.9 4.9 AD-6475961.6 80.3 6.9 5.7 AD-64760 8.7 50.0 3.6 5.7 AD-64761 12.7 36.7 5.7 6.4AD-64762 107.7 98.0 9.1 10.9 AD-64763 72.8 84.8 6.0 9.7 AD-64764 21.757.5 7.8 6.3 AD-64765 67.9 80.2 7.5 3.0 AD-64766 11.3 38.8 5.4 11.3AD-64767 13.2 39.0 5.8 9.5 AD-64768 74.3 102.3 6.5 9.2 AD-64769 57.186.9 5.4 1.1 AD-64770 68.5 78.2 3.2 9.0 AD-64771 45.4 68.5 2.0 8.3AD-64772 11.8 50.0 2.7 1.7 AD-64773 53.8 98.1 1.3 8.6 AD-64774 43.2 82.47.2 8.1 AD-64775 42.9 78.7 1.4 13.8 AD-64776 52.6 70.1 2.9 4.3 AD-6477719.8 65.8 3.7 6.8 AD-64778 7.9 34.7 2.2 9.6 AD-64779 66.9 86.3 7.3 7.9AD-64780 78.8 89.0 7.8 5.1 AD-64781 54.6 82.1 12.9 11.2 AD-64782 9.256.1 5.0 18.3 AD-64783 8.0 35.7 1.4 8.5 AD-64784 52.6 80.3 4.4 6.1AD-64785 92.4 93.9 9.8 4.3 AD-64786 83.4 85.4 8.4 7.5

TABLE 4A Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDname Modified Sense Sequence 5′ to 3′ NOModified Antisense Sequence 5′ to 3′ NO AD-62865UfsgsUfcAfcUfuGfAfAfcUfcAfaCfuCfaAfL96 266usUfsgAfgUfuGfaGfuucAfaGfuGfaCfasusa 278 AD-66916asascuaaCfuAfAfCfuuaauucaaaL96 267 usUfsugaAfuUfAfaguuAfgUfuaguusgsc 279AD-66917 uscsacaaUfuAfAfGfcuccuucuuuL96 268asAfsagaAfgGfAfgcuuAfaUfugugasasc 280 AD-66918gsasgcaaCfuAfAfCfuaacuuaauuL96 269 asAfsuuaAfgUfUfaguuaGfuUfgcucsusu 281AD-66919 ususauugUfuCfCfUfcuaguuauuuL96 270asAfsauaAfcUfAfgaggAfaCfaauaasasa 282 AD-66920asusuaagCfuCfCfUfucuuuuuauuL96 271 asAfsuaaAfaAfGfaaggAfgCfuuaaususg 283AD-66921 usgsucacUfuGfAfAfcucaacucaaL96 272usUfsgagUfuGfAfguucAfaGfugacasusa 284 AD-66922gsasauauGfuCfAfCfuugaacucaaL96 273 usUfsgagUfuCfAfagugAfcAfuauucsusu 285AD-66923 ascsauauUfuGfAfUfcagucuuuuuL96 274asAfsaaaGfaCfUfgaucAfaAfuaugususg 286 AD-66924csasacauAfuUfUfGfaucagucuuuL96 275 asAfsagaCfuGfAfucaaAfuAfuguugsasg 287AD-66925 csusccauAfgUfGfAfagcaaucuaaL96 276usUfsagaUfuGfCfuucaCfuAfuggagsusa 288 AD-65695ascsauauUfuGfAfUfcagucuuuuuL96 277 asAfsaaaGfacugaucAfaAfuaugususg 289

TABLE 4B Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDname Modified Sense Sequence 5′ to 3′ NOModified Antisense Sequence 5′ to 3′ NO AD-62865UfsgsUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf 266usUfsgAfgUfuGfaGfuucAfaGfuGfaCfasusa 278 AD-66916asascuaaCfuAfAfCfuuaauucaaa 267 usUfsugaAfuUfAfaguuAfgUfuaguusgsc 279AD-66917 uscsacaaUfuAfAfGfcuccuucuuu 268asAfsagaAfgGfAfgcuuAfaUfugugasasc 280 AD-66918gsasgcaaCfuAfAfCfuaacuuaauu 269 asAfsuuaAfgUfUfaguuaGfuUfgcucsusu 281AD-66919 ususauugUfuCfCfUfcuaguuauuu 270asAfsauaAfcUfAfgaggAfaCfaauaasasa 282 AD-66920asusuaagCfuCfCfUfucuuuuuauu 271 asAfsuaaAfaAfGfaaggAfgCfuuaaususg 283AD-66921 usgsucacUfuGfAfAfcucaacucaa 272usUfsgagUfuGfAfguucAfaGfugacasusa 284 AD-66922gsasauauGfuCfAfCfuugaacucaa 273 usUfsgagUfuCfAfagugAfcAfuauucsusu 285AD-66923 ascsauauUfuGfAfUfcagucuuuuu 274asAfsaaaGfaCfUfgaucAfaAfuaugususg 286 AD-66924csasacauAfuUfUfGfaucagucuuu 275 asAfsagaCfuGfAfucaaAfuAfuguugsasg 287AD-66925 csusccauAfgUfGfAfagcaaucuaa 276usUfsagaUfuGfCfuucaCfuAfuggagsusa 288 AD-65695ascsauauUfuGfAfUfcagucuuuuu 277 asAfsaaaGfacugaucAfaAfuaugususg 289

TABLE 5 Additional Unmodified ANGPTL3 RNAi Agents. 5′ Position SEQ SEQof Antisense Duplex Unmodified Sense ID Unmodified Antisense IDStrand in name Sequence 5′ to 3′ NO Sequence 5′ to 3′ NO NM_014495.3AD-62865 UGUCACUUGAACUCAACUCAA 290 UUGAGUUGAGUUCAAGUGACAUA 302 425AD-66916 AACUAACUAACUUAAUUCAAA 291 UUUGAAUUAAGUUAGUUAGUUGC 303 509AD-66917 UCACAAUUAAGCUCCUUCUUU 292 AAAGAAGGAGCUUAAUUGUGAAC 304 83AD-66918 GAGCAACUAACUAACUUAAUU 293 AAUUAAGUUAGUUAGUUGCUCUU 305 505AD-66919 UUAUUGUUCCUCUAGUUAUUU 294 AAAUAACUAGAGGAACAAUAAAA 306 104AD-66920 AUUAAGCUCCUUCUUUUUAUU 295 AAUAAAAAGAAGGAGCUUAAUUG 307 88AD-66921 UGUCACUUGAACUCAACUCAA 296 UUGAGUUGAGUUCAAGUGACAUA 308 424AD-66922 GAAUAUGUCACUUGAACUCAA 297 UUGAGUUCAAGUGACAUAUUCUU 309 420AD-66923 ACAUAUUUGAUCAGUCUUUUU 298 AAAAAGACUGAUCAAAUAUGUUG 310 305AD-66924 CAACAUAUUUGAUCAGUCUUU 299 AAAGACUGAUCAAAUAUGUUGAG 311 303AD-66925 CUCCAUAGUGAAGCAAUCUAA 300 UUAGAUUGCUUCACUAUGGAGUA 312 1041AD-65695 ACAUAUUUGAUCAGUCUUUUU 301 AAAAAGACUGAUCAAAUAUGUUG 313 305

Example 4. In Vivo ANGPTL3 Silencing in Wild-Type Mice

The in vivo efficacy and duration of a subset of the additional agentsdescribed above was assessed in wild-type (C57BL/6) mice. Six to eightweek-old female mice were subcutaneoulsy administered a single 3 mg/kgdose of the agents and the level of mouse ANGPTL3 was determined in theserum of the animals pre-dose, day 0, and on days 4, 10, 17, 27, 38, and52 post-dose. Three mice per group were used for these assays. ANGPTL3levels were assayed utilizing an ELISA assay, R&D Systems MouseAngiopoietin-Like 3 Quantikine ELISA Kit (catalog number MANL30).

The results of these assays are provided in FIGS. 6A and 6B. Asdemonstrated in FIG. 6A, all of the agents potently and durably inhibitANGPTL3 expression and reach a nadir at about day 4 post-administrationAs demonstrated in FIG. 6B, at Day post-dose, the level of ANGPTL3 islowest in animals administered AD-63174 and AD-63175 as compared to theother agents assayed.

A second subset of the additional agents described above was alsoassessed in wild-type (C57BL/6) mice. Six to eight week-old female micewere subcutaneoulsy administered either a single 1 mg/kg dose or asingle 3 mg/kg dose of the agents and the level of mouse ANGPTL3 wasdetermined in the serum of the animals pre-dose, day 0, and at days 5,14, 21, 28, and 42 post-dose. (For the groups of animals administeredAD-65695, due to the increased duration of response to this agent ascompared to the response of the other agents tested, the level of mouseANGPTL3 was also determined in the serum of the animals administeredAD-65695 at day 55 post-dose). Three mice per group were used for theseassays. ANGPTL3 levels were assayed utilizing an ELISA assay, asdescribed above.

The results of these assays are provided in FIGS. 7A and 7B anddemonstrate that all of the agents potently and durably inhibit ANGPTL3expression and reach a nadir at about day 5 post-administration (FIG.7A) and that, relative to the other agents assayed, animals administeredAD-65695 had the lowest ANGPTL3 levels at the 1 mg/kg and 3 mg/kg dose(FIG. 7B).

Example 5. In Vivo ANGPTL3 Silencing in Ob/Ob Mice

The in vivo efficacy and duration of a subset of the additional agentsdescribed above was assessed in ob/ob mice. Six to eight week-old femaleob/ob mice were subcutaneoulsy administered a single 3 mg/kg dose of theagents and the level of mouse ANGPTL3 was determined in the serum of theanimals pre-dose, day 0, and at days 5, 13, 24, and 38 post-dose. Fourmice per group were used for these assays. ANGPTL3 levels were assayedutilizing an ELISA assay, as described above.

The results of these assays are provided in FIGS. 8A and 8B anddemonstrate that all of the agents potently and durably inhibit ANGPTL3expression and reach a nadir at about day 5 post-administration (FIG.8A) and that, relative to the other agents assayed, animals administeredAD-63175 or AD-65695 had the highest silencing of ANGPTL3 levels at adose of 3 mg/kg (FIG. 8B).

The dose response of AD-65695 was assayed in ob/ob mice bysubcutaneously administering a single dose of 0.3 mg/kg, 1 mg/kg, 3mg/kg, or 9 mg/kg to the animals and the level of mouse ANGPTL3 wasdetermined in the serum of the animals pre-dose and at days 5 and 13post-dose. ANGPTL3 levels were assayed utilizing an ELISA assay, asdescribed above, and the levels of serum triglycerides (TGs), lowdensity lipoprotein cholesterol (LDLc), high density lipoproteincholesterol (HDLc) and total cholesterol (TC) were also measured usingan Olympus Analyzer.

FIG. 9 demonstrates that at day 5, there is a 58% silencing of ANGPTL3protein in the serum of animals administered a 0.3 mg/kg dose ofAD-65695, 82% silencing of ANGPTL3 protein in the serum of animalsadministered a 1 mg/kg dose of AD-65695, 98% silencing of ANGPTL3protein in the serum of animals administered a 3 mg/kg dose of AD-65695,and 99% silencing of ANGPTL3 protein in the serum of animalsadministered a 9 mg/kg dose of AD-65695.

Shown in FIG. 10A are levels of TGs in ob/ob mice followingadministration of a PBS control or AD-65695; shown in FIG. 10B arelevels of TC measured in ob/ob mice after administration of PBS controlor AD-65695; shown in FIG. 10C are levels of LDLc in ob/ob micefollowing administration of a PBS control or AD-65695; and shown in FIG.10D is the ratio of HDLc to TC in ob/ob mice following administration ofa PBS control or AD-65695. The data demonstrates that administration ofAD-65695 lowers TGs, LDLc, and TC in ob/ob mice, as compared tocontrols, at all doses assessed (an 80% reduction in serum LDLc and TGswas observed using a single 3 mg/kg does of AD_65695).

Example 6. In Vivo ANGPTL3 Silencing in AAV-TBG-ANGPTL3 Mice

To determine the durability of a single dose of a subset of theadditional agents described above to reduce human ANGPTL3 proteinlevels, at pre-dose day −14 wild-type mice (C57BL/6) were infected byintravenous administration of 1×10¹¹ viral particles of anadeno-associated virus 8 (AAV8) vector encoding the human ANGPTL3 gene(coding region) driven by the liver-specific thyroxine-binding globulin(TBG) promoter (AAV8-TBG-ANGPTL3) in 200 μl. At day 0, mice weresubcutaneously administered a single 3 mg/kg of the agents and the levelof ANGPTL3 was determined in the serum of the animals pre-dose and atdays 5, 14, 28, and 42 post-dose (see FIG. 11). The level of mouseANGPTL3 in the serum samples was determined using the ELISA assaydescribed above, and the level of human ANGPTL3 in the serum samples wasdetermined by ELISA assay which utilizes an antibody that detects humanand Cynomologous ANGPTL3 but does not cross-react with mouse ANGPTL3(R&D Systems Human Angiopoietin-Like 3 Quantikine ELISA Kit)]. Serumfrom naïve mice (mice that were not exposed to AAV) served as thenegative control for these assays. Two of the agents administered,AD-57927 and AD-65695, cross-react with mouse, rat, Cynomologous, andhuman (m/r/cy/h) ANGPTL3 mRNA. The other two agents administered,AD-62865 and AD-62866, only cross-react with Cynomologous and human(cy/h) ANGPTL3 mRNA

The levels of human ANGPTL3 protein following administration of theagents or a PBS control are shown in FIG. 12A and the levels of mouseANGPTL3 following administration of the agents or a PBS control areshown in FIG. 12B and, as expected, there was no silencing of mouseANGPTL3 following administration of the cy/h agents, AD-62865 andAD-62866. The results demonstrate that up to an 80% knock-down of humanANGPTL3 is achieved following administration of the agents with ˜60%knock-down of ANGPTL3 sustained for at least 4 weeks. The data alsodemonstrate that there is >90% knock-down of mouse ANGPTL3 followingadministration of the cross-reactive m/r/cy/h agent, AD-65695, with ˜75%knock-down of ANGPTL3 sustained for at least 4 weeks following a single3 mg/kg dose of AD-65695. Furthermore, the data demonstrate that therewas comparable efficacy and duration of ANGPTL3 knock-down followingadministration of the m/r/cy/h agent, AD-65695, and the cy/h agent,AD-62865.

A single dose durability analysis was also performed with AD-65695,AD-62865 and AD-62866. As shown in FIG. 13, AAV8-TBG-ANGPTL3 infectedmice were administered a single, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg dose ofAD-65695, or AAV8-TBG-ANGPTL3 infected mice were administered a single 1mg/kg or 3 mg/kg dose of AD-62865, or AAV8-TBG-ANGPTL3 infected micewere administered a single 0.3 mg/kg dose of AD-62866. Serum wascollected from the animals pre-dose and at days 11, 25, and 39 post-doseand the level of mouse and human ANGPTL3 was determined by ELISA assaysas described above.

The data from these analyses are presented in FIGS. 14A, 14B, 15A, and15B. FIGS. 14A and 14B provide the levels of human and mouse ANGPTL3,respectively, following administration of the agents at day 11post-dose. FIGS. 15A and 15B provide the levels of human and mouseANGPTL3, respectively, following administration of a single, 0.3 mg/kg,1 mg/kg, or 3 mg/kg dose of AD-65695 pre-dose and at days 11, 25, and39. The data demonstrate that AD-65695 (m/r/cy/h agent) has an effectivedose in 80% of the animals (ED₈₀) of 3 mg/kg and an ED₄₀ of 1 mg/kg forhuman ANGPTL3. The data also demonstrate that AD-65695 has an ED₉₀ of 3mg/kg, an ED₇₀ of 1 mg/kg, and an ED₅₀ of 0.3 mg/kg for mouse ANGPTL3.In addition, the data demonstrate that AD-62865 (cy/h agent) has an ED₈₀of 3 mg/kg for human ANGPTL3.

The efficacy of additional agents having fewer 2′-fluoro modifiednucleotides as compared to the parent sequence from which they werederived (described in Tables 4A and 4B above) was assayed inAAV8-TBG-ANGPTL3 infected mice by administration of a single 1 mg/kgdose of the agents. Serum was collected from the animals pre-dose and atday 9 post-dose and the level of mouse and human ANGPTL3 was determinedby ELISA assays as described above. The results of these assays areprovided in Table 6, below, and FIG. 16 which demonstrate that three ofthe agents having fewer 2′-fluoro modified nucleotides as compared tothe parent sequence, AD-66920, AD-66921, and AD-66922, have improvedduration of human ANGPTL3 knock-down over the cy/h specific agent,AD-62865.

TABLE 6 In Vivo ANGPTL3 Silencing in AAV-TBG-ANGPTL3 Mice 5′ TargetPosition of Day 9, Relative to Pre-dose Antisense Duplex Dose HumanAngptl3 Mouse Angptl3 Mouse cross Strand in Group n# Name (mg/kg) MeanSD Mean SD reactivity NM_014495.3 1 2 PBS 0.8 0.3 1.4 0.08 2 3 AD-628651 0.44 0.14 1.0 0.16 3 3 AD-66916 1 0.44 0.11 1.3 0.25 482 4 3 AD-669171 0.58 0.22 1.2 0.17 56 5 3 AD-66918 1 1.39 0.68 0.9 0.42 478 6 3AD-66919 1 0.88 0.25 1.1 0.16 77 7 3 AD-66920 1 0.41 0.05 1.0 0.14 61 83 AD-66921 1 0.39 0.14 1.1 0.40 398 9 3 AD-66922 1 0.27 0.08 1.2 0.38393 10 3 AD-66923 1 0.67 0.21 0.3 0.07 yes 278 11 3 AD-66924 1 0.62 0.200.5 0.07 yes 276 12 3 AD-66925 1 0.70 0.22 0.9 0.11 1014 3 AD-65695 10.60 0.14 0.3 0.08 yes 278

TABLE 7A Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDname Modified Sense Sequence 5′ to 3′ NOModified Antisense Sequence 5′ to 3′ NO AD-67021gsasauadTgudGacuugaa(Cgn)ucaaL96 314 usdTsgaguucaagdTgdAcauauucsusu 335AD-67022 gsasauadTgudGacuugaa(Cgn)ucaaL96 315us(Ufms)gaguucaagdTgdAcauauucsusu 336 AD-67173gsasauauGfuCfAfCfuugaacucaaL96 316 usdTsgaguucaagdTgdAcauauucsusu 337AD-67174 gsasauadTgudGacuugaa(Cgn)ucaaL96 317usUfsgagUfuCfAfagugAfcAfuauucsusu 338 AD-66922gsasauauGfuCfAfCfuugaacucaaL96 318 usUfsgagUfuCfAfagugAfcAfuauucsusu 339AD-67008 asusuaadGcudGcuucuuu(Tgn)uauuL96 319asdAsuaaaaagaadGgdAgcuuaaususg 340 AD-67007asusuaadGcudGcuucuuu(Tgn)uauuL96 320 asAfsuaaAfaagaaggAfgCfuuaaususg 341AD-67006 asusuaagCfuCfCfUfucuuuuuauuL96 321asdAsuaaaaagaadGgdAgcuuaaususg 342 AD-66920asusuaagCfuCfCfUfucuuuuuauuL96 322 asAfsuaaAfaAfGfaaggAfgCfuuaaususg 343AD-67028 ascsauadTuudCaucaguc(Tgn)uuuuL96 323asdAsaaagacugadTcdAaauaugususg 344 AD-67013usgsucadCuudCaacucaa(Cgn)ucaaL96 324 usdTsgaguugagudTcdAagugacasusa 345AD-67014 usgsucadCuudCaacucaa(Cgn)ucaaL96 325us(Ufms)gaguugagudTcdAagugacasusa 346 AD-66921usgsucacUfuGfAfAfcucaacucaaL96 326 usUfsgagUfuGfAfguucAfaGfugacasusa 347AD-66974 asascuaacuuacuuaau(Tgn)caaaL96 327usdTsugaauuaagdTudAguuaguusgsc 348 AD-66973asascudAacuuacuuaau(Tgn)caaaL96 328 usdTsugaauuaagdTudAguuaguusgsc 349AD-66916 asascuaaCfuAfAfCfuuaauucaaaL96 329usUfsugaAfuUfAfaguuAfgUfuaguusgsc 350 AD-66925csusccauAfgUfGfAfagcaaucuaaL96 330 usUfsagaUfuGfCfuucaCfuAfuggagsusa 351AD-67042 csusccadTagdAgaagcaa(Tgn)cuaaL96 331usdTsagauugcuudCadCuauggagsusa 352 AD-66923ascsauauUfuGfAfUfcagucuuuuuL96 332 asAfsaaaGfaCfUfgaucAfaAfuaugususg 353AD-67026 ascsauauUfuGfAfUfcagucuuuuuL96 333asdAsaaagacugadTcdAaauaugususg 354 AD-67027ascsauadTuudCaucaguc(Tgn)uuuuL96 334 asAfsaaaGfacugaucAfaAfuaugususg 355

TABLE 7B Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDname Modified Sense Sequence 5′ to 3′ NOModified Antisense Sequence 5′ to 3′ NO AD-67021gsasauadTgudGacuugaa(Cgn)ucaa 314 usdTsgaguucaagdTgdAcauauucsusu 335AD-67022 gsasauadTgudGacuugaa(Cgn)ucaa 315us(Ufms)gaguucaagdTgdAcauauucsusu 336 AD-67173gsasauauGfuCfAfCfuugaacucaa 316 usdTsgaguucaagdTgdAcauauucsusu 337AD-67174 gsasauadTgudGacuugaa(Cgn)ucaa 317usUfsgagUfuCfAfagugAfcAfuauucsusu 338 AD-66922gsasauauGfuCfAfCfuugaacucaa 318 usUfsgagUfuCfAfagugAfcAfuauucsusu 339AD-67008 asusuaadGcudGcuucuuu(Tgn)uauu 319asdAsuaaaaagaadGgdAgcuuaaususg 340 AD-67007asusuaadGcudGcuucuuu(Tgn)uauu 320 asAfsuaaAfaagaaggAfgCfuuaaususg 341AD-67006 asusuaagCfuCfCfUfucuuuuuauu 321 asdAsuaaaaagaadGgdAgcuuaaususg342 AD-66920 asusuaagCfuCfCfUfucuuuuuauu 322asAfsuaaAfaAfGfaaggAfgCfuuaaususg 343 AD-67028ascsauadTuudCaucaguc(Tgn)uuuu 323 asdAsaaagacugadTcdAaauaugususg 344AD-67013 usgsucadCuudCaacucaa(Cgn)ucaa 324usdTsgaguugagudTcdAagugacasusa 345 AD-67014usgsucadCuudCaacucaa(Cgn)ucaa 325 us(Ufms)gaguugagudTcdAagugacasusa 346AD-66921 usgsucacUfuGfAfAfcucaacucaa 326usUfsgagUfuGfAfguucAfaGfugacasusa 347 AD-66974asascuaacuuacuuaau(Tgn)caaa 327 usdTsugaauuaagdTudAguuaguusgsc 348AD-66973 asascudAacuuacuuaau(Tgn)caaa 328 usdTsugaauuaagdTudAguuaguusgsc349 AD-66916 asascuaaCfuAfAfCfuuaauucaaa 329usUfsugaAfuUfAfaguuAfgUfuaguusgsc 350 AD-66925csusccauAfgUfGfAfagcaaucuaa 330 usUfsagaUfuGfCfuucaCfuAfuggagsusa 351AD-67042 csusccadTagdAgaagcaa(Tgn)cuaa 331usdTsagauugcuudCadCuauggagsusa 352 AD-66923 ascsauauUfuGfAfUfcagucuuuuu332 asAfsaaaGfaCfUfgaucAfaAfuaugususg 353 AD-67026ascsauauUfuGfAfUfcagucuuuuu 333 asdAsaaagacugadTcdAaauaugususg 354AD-67027 ascsauadTuudCaucaguc(Tgn)uuuu 334asAfsaaaGfacugaucAfaAfuaugususg 355

TABLE 7C Additional Unmodified ANGPTL3 RNAi Agents. DuplexModified Sense SEQ ID Modified Antisense SEQ ID name Sequence 5′ to 3′NO Sequence 5′ to 3′ NO AD-67021 GAAUATGUGACUUGAACUCAA 356UTGAGUUCAAGTGACAUAUUCUU 377 AD-67022 GAAUATGUGACUUGAACUCAA 357UUGAGUUCAAGTGACAUAUUCUU 378 AD-67173 GAAUAUGUCACUUGAACUCAA 358UTGAGUUCAAGTGACAUAUUCUU 379 AD-67174 GAAUATGUGACUUGAACUCAA 359UUGAGUUCAAGUGACAUAUUCUU 380 AD-66922 GAAUAUGUCACUUGAACUCAA 360UUGAGUUCAAGUGACAUAUUCUU 381 AD-67008 AUUAAGCUGCUUCUUUTUAUU 361AAUAAAAAGAAGGAGCUUAAUUG 382 AD-67007 AUUAAGCUGCUUCUUUTUAUU 362AAUAAAAAGAAGGAGCUUAAUUG 383 AD-67006 AUUAAGCUCCUUCUUUUUAUU 363AAUAAAAAGAAGGAGCUUAAUUG 384 AD-66920 AUUAAGCUCCUUCUUUUUAUU 364AAUAAAAAGAAGGAGCUUAAUUG 385 AD-67028 ACAUATUUCAUCAGUCTUUUU 365AAAAAGACUGATCAAAUAUGUUG 386 AD-67013 UGUCACUUCAACUCAACUCAA 366UTGAGUUGAGUTCAAGUGACAUA 387 AD-67014 UGUCACUUCAACUCAACUCAA 367UUGAGUUGAGUTCAAGUGACAUA 388 AD-66921 UGUCACUUGAACUCAACUCAA 368UUGAGUUGAGUUCAAGUGACAUA 389 AD-66974 AACUAACUUACUUAAUTCAAA 369UTUGAAUUAAGTUAGUUAGUUGC 390 AD-66973 AACUAACUUACUUAAUTCAAA 370UTUGAAUUAAGTUAGUUAGUUGC 391 AD-66916 AACUAACUAACUUAAUUCAAA 371UUUGAAUUAAGUUAGUUAGUUGC 392 AD-66925 CUCCAUAGUGAAGCAAUCUAA 372UUAGAUUGCUUCACUAUGGAGUA 393 AD-67042 CUCCATAGAGAAGCAATCUAA 373UTAGAUUGCUUCACUAUGGAGUA 394 AD-66923 ACAUAUUUGAUCAGUCUUUUU 374AAAAAGACUGAUCAAAUAUGUUG 395 AD-67026 ACAUAUUUGAUCAGUCUUUUU 375AAAAAGACUGATCAAAUAUGUUG 396 AD-67027 ACAUATUUCAUCAGUCTUUUU 376AAAAAGACUGAUCAAAUAUGUUG 397

Example 7. In Vivo ANGPTL3 Silencing in AAV-TBG-ANGPTL3 Mice

A second subset of the additional agents listed in Tables 7A, 7B, and 7Cwere assayed in AAV8-TBG-ANGPTL3 infected mice. The modified nucleotidesequences of the sense and antisense strands of these sequences areprovided in Tables 8A and 8B. Briefly, and as described above, atpre-dose day −14, wild-type mice (C57BL/6) were infected by intravenousadministration of 1×10¹¹ viral particles. At day 0, mice wereadministered a single 1 mg/kg or 3 mg/kg dose of the agents. Serum wascollected from the animals pre-dose and at days 14 and 28 post-dose andthe level of mouse and human ANGPTL3 was determined by ELISA assays asdescribed above.

The results of these assays are provided in Table 9, below, and FIGS.17A and 17B. The results demonstrate that agents AD-67173, AD-67174,AD-66922, AD-67007, and AD-66920 have improved duration of human ANGPTL3knock-down.

TABLE 8A SEQ SEQ ID ID Duplex ID Sense Sequence 5′ to 3′ NOAntisense Sequence 5′ to 3′ NO AD-67021 gsasauadTgudGacuugaa(Cgn)ucaaL96398 usdTsgaguucaagdTgdAcauauucsusu 408 AD-67022gsasauadTgudGacuugaa(Cgn)ucaaL96 399 us(Ufms)gaguucaagdTgdAcauauucsusu409 AD-67173 gsasauauGfuCfAfCfuugaacucaaL96 400usdTsgaguucaagdTgdAcauauucsusu 410 AD-67174gsasauadTgudGacuugaa(Cgn)ucaaL96 401 usUfsgagUfuCfAfagugAfcAfuauucsusu411 AD-66922 gsasauauGfuCfAfCfuugaacucaaL96 402usUfsgagUfuCfAfagugAfcAfuauucsusu 412 AD-67008asusuaadGcudGcuucuuu(Tgn)uauuL96 403 asdAsuaaaaagaadGgdAgcuuaaususg 413AD-67007 asusuaadGcudGcuucuuu(Tgn)uauuL96 404asAfsuaaAfaagaaggAfgCfuuaaususg 414 AD-67006asusuaagCfuCfCfUfucuuuuuauuL96 405 asdAsuaaaaagaadGgdAgcuuaaususg 415AD-66920 asusuaagCfuCfCfUfucuuuuuauuL96 406asAfsuaaAfaAfGfaaggAfgCfuuaaususg 416 AD-67028ascsauadTuudCaucaguc(Tgn)uuuuL96 407 asdAsaaagacugadTcdAaauaugususg 417

TABLE 8B SEQ SEQ ID ID Duplex ID Sense Sequence 5′ to 3′ NOAntisense Sequence 5′ to 3′ NO AD-67021 gsasauadTgudGacuugaa(Cgn)ucaa398 usdTsgaguucaagdTgdAcauauucsusu 408 AD-67022gsasauadTgudGacuugaa(Cgn)ucaa 399 us(Ufms)gaguucaagdTgdAcauauucsusu 409AD-67173 gsasauauGfuCfAfCfuugaacucaa 400 usdTsgaguucaagdTgdAcauauucsusu410 AD-67174 gsasauadTgudGacuugaa(Cgn)ucaa 401usUfsgagUfuCfAfagugAfcAfuauucsusu 411 AD-66922gsasauauGfuCfAfCfuugaacucaa 402 usUfsgagUfuCfAfagugAfcAfuauucsusu 412AD-67008 asusuaadGcudGcuucuuu(Tgn)uauu 403asdAsuaaaaagaadGgdAgcuuaaususg 413 AD-67007asusuaadGcudGcuucuuu(Tgn)uauu 404 asAfsuaaAfaagaaggAfgCfuuaaususg 414AD-67006 asusuaagCfuCfCfUfucuuuuuauu 405 asdAsuaaaaagaadGgdAgcuuaaususg415 AD-66920 asusuaagCfuCfCfUfucuuuuuauu 406asAfsuaaAfaAfGfaaggAfgCfuuaaususg 416 AD-67028ascsauadTuudCaucaguc(Tgn)uuuu 407 asdAsaaagacugadTcdAaauaugususg 417

TABLE 9 Day 14, Relative to Pre-dose Dose Human Angptl3 Duplex (mg/kg)Mean SD PBS 0 0.74 0.27 AD-67021 3 0.48 0.22 AD-67022 3 0.52 0.17AD-67173 3 0.25 0.03 AD-67174 3 0.22 0.05 AD-66922 1 0.26 0.06 AD-670083 0.50 0.18 AD-67007 3 0.21 0.02 AD-67006 3 0.50 0.18 AD-66920 1 0.250.05 AD-65695 1 0.49 0.15 AD-67028 3 0.47 0.04

In another experiment, a further subset of the additional agents listedin Tables 7A, 7B, and 7C were assayed in AAV8-TBG-ANGPTL3 infected mice.The modified nucleotide sequences of the sense and antisense strands ofthese sequences are provided in Tables 10A and 10B. Briefly, and asdescribed above, at pre-dose day −14, wild-type mice (C57BL/6) wereinfected by intravenous administration of 1×10¹¹ viral particles. At day0, mice were administered a single 1 mg/kg or 3 mg/kg dose of theagents. Serum was collected from the animals pre-dose and at days 14 and28 post-dose and the level of mouse and human ANGPTL3 was determined byELISA assays as described above.

The results of these assays at day 14 post-dose are provided in Table11, below, and FIG. 18A, and the results of these assays at day 28post-dose are provided in Table 12, below and FIG. 18B. The resultsdemonstrate that agents AD-66921, AD-66916, and AD-67042 have improvedduration of human ANGPTL3 knock-down.

TABLE 10A SEQ SEQ ID ID Duplex ID Sense Sequene 5′ to 3′ NOAntisense Sequence 5′ to 3′ NO AD-67013 usgsucadCuudCaacucaa(Cgn)ucaaL96418 usdTsgaguugagudTcdAagugacasusa 430 AD-67014usgsucadCuudCaacucaa(Cgn)ucaaL96 419 us(Ufms)gaguugagudTcdAagugacasusa431 AD-66921 usgsucacUfuGfAfAfcucaacucaaL96 420usUfsgagUfuGfAfguucAfaGfugacasusa 432 AD-66974asascuaacuuacuuaau(Tgn)caaaL96 421 usdTsugaauuaagdTudAguuaguusgsc 433AD-66973 asascudAacuuacuuaau(Tgn)caaaL96 422usdTsugaauuaagdTudAguuaguusgsc 434 AD-66916asascuaaCfuAfAfCfuuaauucaaaL96 423 usUfsugaAfuUfAfaguuAfgUfuaguusgsc 435AD-66925 csusccauAfgUfGfAfagcaaucuaaL96 424usUfsagaUfuGfCfuucaCfuAfuggagsusa 436 AD-67042csusccadTagdAgaagcaa(Tgn)cuaaL96 425 usdTsagauugcuudCadCuauggagsusa 437AD-66923 ascsauauUfuGfAfUfcagucuuuuuL96 426asAfsaaaGfaCfUfgaucAfaAfuaugususg 438 AD-67026ascsauauUfuGfAfUfcagucuuuuuL96 427 asdAsaaagacugadTcdAaauaugususg 439AD-67027 ascsauadTuudCaucaguc(Tgn)uuuuL96 428asAfsaaaGfacugaucAfaAfuaugususg 440 AD-67028ascsauadTuudCaucaguc(Tgn)uuuuL96 429 asdAsaaagacugadTcdAaauaugususg 441

TABLE 10B SEQ SEQ ID ID Duplex ID Sense Sequene 5′ to 3′ NOAntisense Sequence 5′ to 3′ NO AD-67013 usgsucadCuudCaacucaa(Cgn)ucaa418 usdTsgaguugagudTcdAagugacasusa 430 AD-67014usgsucadCuudCaacucaa(Cgn)ucaa 419 us(Ufms)gaguugagudTcdAagugacasusa 431AD-66921 usgsucacUfuGfAfAfcucaacucaa 420usUfsgagUfuGfAfguucAfaGfugacasusa 432 AD-66974asascuaacuuacuuaau(Tgn)caaa 421 usdTsugaauuaagdTudAguuaguusgsc 433AD-66973 asascudAacuuacuuaau(Tgn)caaa 422 usdTsugaauuaagdTudAguuaguusgsc434 AD-66916 asascuaaCfuAfAfCfuuaauucaaa 423usUfsugaAfuUfAfaguuAfgUfuaguusgsc 435 AD-66925csusccauAfgUfGfAfagcaaucuaa 424 usUfsagaUfuGfCfuucaCfuAfuggagsusa 436AD-67042 csusccadTagdAgaagcaa(Tgn)cuaa 425usdTsagauugcuudCadCuauggagsusa 437 AD-66923 ascsauauUfuGfAfUfcagucuuuuu426 asAfsaaaGfaCfUfgaucAfaAfuaugususg 438 AD-67026ascsauauUfuGfAfUfcagucuuuuu 427 asdAsaaagacugadTcdAaauaugususg 439AD-67027 ascsauadTuudCaucaguc(Tgn)uuuu 428asAfsaaaGfacugaucAfaAfuaugususg 440 AD-67028ascsauadTuudCaucaguc(Tgn)uuuu 429 asdAsaaagacugadTcdAaauaugususg 441

TABLE 11 Day 14, Relative to PBS Dose Human Angptl3 siRNA (mg/kg) MeanSD PBS 0 1.00 0.12 AD-67013 3 0.86 0.06 AD-67014 3 0.67 0.15 AD-66921 10.44 0.18 AD-66974 3 0.75 0.03 AD-66973 3 0.76 0.05 AD-66916 1 0.41 0.05AD-66925 1 0.65 0.13 AD-67042 3 AD-65695 1 0.59 0.19 AD-66923 1 0.640.25 AD-67026 3 0.56 0.03 AD-67027 3 0.66 0.23 AD-67028 3 0.65 0.21

TABLE 12 Day 28, Relative to PBS Dose Human Angptl3 siRNA (mg/kg) MeanSD PBS 0 1.00 0.12 AD-67013 3 1.50 1.28 AD-67014 3 0.90 0.23 AD-66921 10.44 0.11 AD-66974 3 0.96 0.10 AD-66973 3 1.00 0.34 AD-66916 1 0.57 0.06AD-66925 1 1.01 0.28 AD-67042 3 AD-65695 1 0.85 0.17 AD-66923 1 0.850.25 AD-67026 3 0.64 0.06 AD-67027 3 0.58 0.11 AD-67028 3 1.32 1.01

Example 8. In Vivo ANGPTL3 Silencing in Non-Human Primates

Male cynomolgus monkeys (n=3/group) were subcutaneously administered asingle 1 mg/kg dose of AD-65695 or AD-66920, or a single 3 mg/kg dose ofAD-66920 on day 1. Fasted serum was collected on days 4, 8, 11, 15, and22 post-dose and the protein level of Angplt3 and the serum lipid levelswere measured.

The results of these assays are provided in FIGS. 19A and 19B anddemonstrate that there is up to an 80% knockdown of serum ANGPTL3protein following a single 3 mg/kg dose of AD-66920 (h/cy) and about a50% lowering of the level of serum triglycerides. A single 1 mg/kg doseof AD-66920 had a modest effect on the level of serum triglycerides.Surprisingly, although administration of a single 1 mg/kg dose ofAD-65695 lowered ANGPTL3 protein levels to a similar level as a single 1mg/kg dose of AD-66920, a single 1 mg/kg dose of AD-65695 was aboutthree times less potent than a single 1 mg/kg dose of AD-66920 in theAAV8-TBG-ANGPTL3 mouse model and had no effect on serum triglycerides.

Example 9. In Vivo ANGPTL3 Silencing in AAV-TBG-ANGPTL3 Mice

The in vivo efficacy of an additional set of agents targeting ANGPTL3was assayed in AAV8-TBG-ANGPTL3 infected mice. The modified nucleotidesequences of the sense and antisense strands of these sequences areprovided in Tables 13A and 13B. Briefly, and as described above, atpre-dose day −21 wild-type mice (C57BL/6; n=3) were infected byintravenous administration of 1×10¹¹ viral particles. At day 0, micewere administered a single 1 mg/kg dose of the agents. Serum wascollected from the animals pre-dose and at day 28 post-dose and thelevel of human ANGPTL3 was determined by ELISA assays as describedabove.

The results of these assays are provided in FIG. 20 and demonstrate thatthese agents effectively knockdown human ANGPTL3 protein levels.

A further set of the agents targeting ANGPTL3 listed in Tables 13A and13B was assayed for in vivo efficacy in AAV8-TBG-ANGPTL3 infected mice.As described above, at pre-dose day −21 wild-type mice (C57BL/6; n=3)were infected by intravenous administration of 1×10¹¹ viral particles.At day 0, mice were administered a single 1 mg/kg dose of the agents.Serum was collected from the animals pre-dose and at day 28 post-doseand the level of human and mouse ANGPTL3 was determined by ELISA assaysas described above.

The results of these assays are provided in FIGS. 21A and 21B anddemonstrate that these agents effectively knockdown mouse and humanANGPTL3 protein levels.

Example 10. In Vitro ANGPTL3 Silencing in AAV-TBG-ANGPTL3 Mice

Further iRNA agents targeting the ANGPTL3 gene were synthesized asdescribed above. A detailed list of the additional unmodified ANGPTL3sense and antisense strand sequences is shown in Table 14 and a detailedlist of the modified sense and antisense strand sequences of theseadditional agents is shown in Tables 13A, 13B, 15A and 15B.

These additional agents were evaluated in in vitro assays in Hep3b andprimary Cynomolgus monkey hepatocytes by single dose transfections ofthe agents at 10 nM and 0.1 nM final duplex concentration. (See Table16).

Hep3b cells were cultured and transfected as described above. Freeuptake silencing in primary cynomolgus hepatocytes was assessedfollowing incubation with ANGPTL3 agents for 24 hours. The method wassimilar to that described above, with the exception that 5 μL completegrowth medium was substituted for the 5 μL containing LipofectamineRNAiMax and Optimem. The results of these assays (provided in Table 16)illustrate that many of the duplexes potently inhibited ANGPTL3 mRNAexpression.

TABLE 13A Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDName Sense Sequence 5′ to 3′ NO Antisense Sequence 5′ to 3′ NO AD-66920asusuaagCfuCfCfUfucuuuuuauuL96 442 asAfsuaaAfaAfGfaaggAfgCfuuaaususg 454AD-72868 asusuaagCfuCfCfUfucuuuuuauaL96 443VP(Tams)AfsuaaAfaAfGfaaggAfgCfuuaaususg 455 AD-72869asusuaagcuCfcUfucuuuuuauuL96 444 asAfsuaaaaagaaGfgAfgcuuaaususg 456AD-72870 asusuaagcuCfcUfucuuuuuauuL96 445 asAfsuaaaaagaadGgAfgcuuaaususg457 AD-72871 asusuaagcuCfcUfucuuu(Tgn)uauuL96 446asAfsuaaaaagaaGfgAfgcuuaaususg 458 AD-72872 asusuaagcuCfcUfucuauauauuL96447 asAfsuaaaaagaaGfgAfgcuuaaususg 459 AD-65695ascsauauUfuGfAfUfcagucuuuuuL96 448 asAfsaaaGfacugaucAfaAfuaugususg 460AD-72873 ascsauauUfuGfAfUfcagucuuuuaL96 449VP(Tams)AfsaaaGfacugaucAfaAfuaugususg 461 AD-72874ascsauauuuGfaUfcagucuuuuuL96 450 asAfsaaagacugaUfcAfaauaugususg 462AD-72875 ascsauauuuGfaUfcagucuuuuuL96 451 asAfsaaagacugadTcAfaauaugususg463 AD-72876 ascsauauuuGfaUfcaguc(Tgn)uuuuL96 452asAfsaaagacugaUfcAfaauaugususg 464 AD-72877 ascsauauuuGfaUfcagacauuuuL96453 asAfsaaagacugaUfcAfaauaugususg 465

TABLE 13B Additional Modified ANGPTL3 RNAi Agents. Duplex SEQ ID SEQ IDName Sense Sequence 5′ to 3′ NO Antisense Sequence 5′ to 3′ NO AD-66920asusuaagCfuCfCfUfucuuuuuauu 442 asAfsuaaAfaAfGfaaggAfgCfuuaaususg 454AD-72868 asusuaagCfuCfCfUfucuuuuuaua 443VP(Tams)AfsuaaAfaAfGfaaggAfgCfuuaaususg 455 AD-72869asusuaagcuCfcUfucuuuuuauu 444 asAfsuaaaaagaaGfgAfgcuuaaususg 456AD-72870 asusuaagcuCfcUfucuuuuuauu 445 asAfsuaaaaagaadGgAfgcuuaaususg457 AD-72871 asusuaagcuCfcUfucuuu(Tgn)uauu 446asAfsuaaaaagaaGfgAfgcuuaaususg 458 AD-72872 asusuaagcuCfcUfucuauauauu447 asAfsuaaaaagaaGfgAfgcuuaaususg 459 AD-65695ascsauauUfuGfAfUfcagucuuuuu 448 asAfsaaaGfacugaucAfaAfuaugususg 460AD-72873 ascsauauUfuGfAfUfcagucuuuua 449VP(Tams)AfsaaaGfacugaucAfaAfuaugususg 461 AD-72874ascsauauuuGfaUfcagucuuuuu 450 asAfsaaagacugaUfcAfaauaugususg 462AD-72875 ascsauauuuGfaUfcagucuuuuu 451 asAfsaaagacugadTcAfaauaugususg463 AD-72876 ascsauauuuGfaUfcaguc(Tgn)uuuu 452asAfsaaagacugaUfcAfaauaugususg 464 AD-72877 ascsauauuuGfaUfcagacauuuu453 asAfsaaagacugaUfcAfaauaugususg 465

TABLE 14 Additional Unmodified ANGPTL3 RNAi Agents. SEQ SEQAntisense target Duplex ID Antisense ID position in NameSense Sequence 5′ to 3′ NO Sequence 5′ to 3′ NO NM_014495.3 AD-66916AACUAACUAACUUAAUUCAAA 466 UUUGAAUUAAGUUAGUUAGUUGC 507 480-502 AD-66922GAAUAUGUCACUUGAACUCAA 467 UUGAGUUCAAGUGACAUAUUCUU 508 391-413 AD-74755AAACUCUAAACUUGACUAAAU 468 AUUUAGUCAAGUUUAGAGUUUUA 509 1853-1874 AD-74756AACUAACUUAAUUCAAAAUCA 469 UGAUUUUGAAUUAAGUUAGUUAG 510 515-536 AD-74757CCAGAAGUAACUUCACUUAAA 470 UUUAAGUGAAGUUACUUCUGGGU 511 555-576 AD-74758GAACUCAACUCAAAACUUGAA 471 UUCAAGUUUUGAGUUGAGUUCAA 512 435-456 AD-74759CAACUCAAAACUUGAAAGCCU 472 AGGCUUUCAAGUUUUGAGUUGAG 513 440-461 AD-74760UUCCACGUUGCUUGAAAUUGA 473 UCAAUUUCAAGCAACGUGGAACU 514 46-67 AD-74761GUUGCUUGAAAUUGAAAAUCA 474 UGAUUUUCAAUUUCAAGCAACGU 515 52-73 AD-74762GAUCACAAAACUUCAAUGAAA 475 UUUCAUUGAAGUUUUGUGAUCCA 516 952-973 AD-74763UCAAGAUUUGCUAUGUUAGAA 476 UUCUAACAUAGCAAAUCUUGAUU 517 183-204_C21AAD-74764 CAAAAUCAAGAUUUGCUAUGU 477 ACAUAGCAAAUCUUGAUUUUGGC 518 178-199AD-74765 GAACUACUCCCUUUCUUCAGU 478 ACUGAAGAAAGGGAGUAGUUCUU 519 751-772AD-74766 AGAAAUUUCUCUAUCUUCCAA 479 UUGGAAGAUAGAGAAAUUUCUGU 520 716-737AD-74767 UGAACUGAGGCAAAUUUAAAA 480 UUUUAAAUUUGCCUCAGUUCAUU 521 1461-1482AD-74768 CAUCCAACAGAUUCAGAAAGA 481 UCUUUCUGAAUCUGUUGGAUGGA 5221434-1455_C21A AD-74769 AAAAUCAAGAUUUGCUAUGUU 482AACAUAGCAAAUCUUGAUUUUGG 523 179-200 AD-74770 AGAGCAAAAUCUAAGCCAGAA 483UUCUGGCUUAGAUUUUGCUCUUG 524 1341-1362_G21A AD-74771AUCAUAUGAGCUAAUAUCACA 484 UGUGAUAUUAGCUCAUAUGAUGC 525 1798-1819 AD-74772AAUAAACCUCGUAACAAGUUA 485 UAACUUGUUACGAGGUUUAUUUC 526 2339-2360 AD-74773CAACAGCAUAGUCAAAUAAAA 486 UUUUAUUUGACUAUGCUGUUGGU 527 651-672 AD-74774AAAACAACCUAAAUGGUAAAU 487 AUUUACCAUUUAGGUUGUUUUCU 528 1309-1330 AD-74775CACUUAAAACUUUUGUAGAAA 488 UUUCUACAAAAGUUUUAAGUGAA 529 568-589 AD-74776AUUAAAAUAAGUUCGCUGUCU 489 AGACAGCGAACUUAUUUUAAUAC 530 2070-2091 AD-74777CAAAACUUCAAUGAAACGUGA 490 UCACGUUUCAUUGAAGUUUUGUG 531 957-978_G21AAD-74778 AGAUGGAUCACAAAACUUCAA 491 UUGAAGUUUUGUGAUCCAUCUAU 532 947-968AD-74779 CACUUAAUACUAUGAAAACAA 492 UUGUUUUCAUAGUAUUAAGUGUU 533 2194-2215AD-74780 ACUAAGUCACAUUGACUUUAA 493 UUAAAGUCAAUGUGACUUAGUAG 534 2111-2132AD-74781 UAUUAAAUAACUUUUCUAAAU 494 AUUUAGAAAAGUUAUUUAAUAAG 535 1746-1767AD-74782 ACACUUAAUACUAUGAAAACA 495 UGUUUUCAUAGUAUUAAGUGUUA 536 2193-2214AD-74783 UUUAUGAAACCUAAUGAAGCA 496 UGCUUCAUUAGGUUUCAUAAAUA 537 2035-2056AD-74784 CAAACAUUAUAUUGAAUAUUA 497 UAAUAUUCAAUAUAAUGUUUGUU 5381103-1124_C21A AD-74785 AAACCAGUGAAAUCAAAGAAA 498UUUCUUUGAUUUCACUGGUUUGC 539 343-364_G21A AD-74786 GAGUUAAAGUUUAUAUUUCCA499 UGGAAAUAUAAACUUUAACUCGA 540 2271-2292_C21A AD-74787CCAAUAUAAACAAUUAAACCA 500 UGGUUUAAUUGUUUAUAUUGGUC 541 632-653 AD-74788GUGGAGAAAACAACCUAAAUA 501 UAUUUAGGUUGUUUUCUCCACAC 542 1303-1324_G21AAD-74789 AACUCAACUCAAAACUUGAAA 502 UUUCAAGUUUUGAGUUGAGUUCA 543 436-457AD-74790 AAUGUUCACAAUUAAGCUCCU 503 AGGAGCUUAAUUGUGAACAUUUU 544  80-101AD-74791 GCAGAAUUAAAUACUGUAUUA 504 UAAUACAGUAUUUAAUUCUGCUU 545 2053-2074AD-74792 UGAAUGAAAUAAGAAAUGUAA 505 UUACAUUUCUUAUUUCAUUCAAC 546 772-793AD-74793 UACAUAUAAACUACAAGUCAA 506 UUGACUUGUAGUUUAUAUGUAGU 547 386-407

TABLE 15A Additional Modified ANGPTL3 RNAi Agents. SEQ SEQ SEQ DuplexSense ID ID mRNA Target Sequence ID Name Sequence 5′ to 3′ NO:Antisense Sequence 5′ to 3′ NO: in NM_014495.3 NO: AD-66916asascuaaCfuAfAf 548 usUfsugaAfuUfAfaguuAfgUfuaguusgsc 589GCAACUAACUAACUUAAUUCAAA 630 CfuuaauucaaaL96 AD-66922 gsasauauGfuCfAf 549usUfsgagUfuCfAfagugAfcAfuauucsusu 590 AAGAAUAUGUCACUUGAACUCAA 631CfuugaacucaaL96 AD-74755 asasacucUfaAfAf 550asUfsuuaGfuCfAfaguuUfaGfaguuususa 591 UAAAACUCUAAACUUGACUAAAU 632CfuugacuaaauL96 AD-74756 asascuaaCfuUfAf 551usGfsauuUfuGfAfauuaAfgUfuaguusasg 592 CUAACUAACUUAAUUCAAAAUCA 633AfuucaaaaucaL96 AD-74757 cscsagaaGfuAfAf 552usUfsuaaGfuGfAfaguuAfcUfucuggsgsu 593 ACCCAGAAGUAACUUCACUUAAA 634CfuucacuuaaaL96 AD-74758 gsasacucAfaCfUf 553usUfscaaGfuUfUfugagUfuGfaguucsasa 594 UUGAACUCAACUCAAAACUUGAA 635CfaaaacuugaaL96 AD-74759 csasacucAfaAfAf 554asGfsgcuUfuCfAfaguuUfuGfaguugsasg 595 CUCAACUCAAAACUUGAAAGCCU 636CfuugaaagccuL96 AD-74760 ususccacGfuUfGf 555usCfsaauUfuCfAfagcaAfcGfuggaascsu 596 AGUUCCACGUUGCUUGAAAUUGA 637CfuugaaauugaL96 AD-74761 gsusugcuUfgAfAf 556usGfsauuUfuCfAfauuuCfaAfgcaacsgsu 597 ACGUUGCUUGAAAUUGAAAAUCA 638AfuugaaaaucaL96 AD-74762 gsasucacAfaAfAf 557usUfsucaUfuGfAfaguuUfuGfugaucscsa 598 UGGAUCACAAAACUUCAAUGAAA 639CfuucaaugaaaL96 AD-74763 uscsaagaUfuUfGf 558usUfscuaAfcAfUfagcaAfaUfcuugasusu 599 AAUCAAGAUUUGCUAUGUUAGAC 640CfuauguuagaaL96 AD-74764 csasaaauCfaAfGf 559asCfsauaGfcAfAfaucuUfgAfuuuugsgsc 600 GCCAAAAUCAAGAUUUGCUAUGU 641AfuuugcuauguL96 AD-74765 gsasacuaCfuCfCf 560asCfsugaAfgAfAfagggAfgUfaguucsusu 601 AAGAACUACUCCCUUUCUUCAGU 642CfuuucuucaguL96 AD-74766 asgsaaauUfuCfUf 561usUfsggaAfgAfUfagagAfaAfuuucusgsu 602 ACAGAAAUUUCUCUAUCUUCCAA 643CfuaucuuccaaL96 AD-74767 usgsaacuGfaGfGf 562usUfsuuaAfaUfUfugccUfcAfguucasusu 603 AAUGAACUGAGGCAAAUUUAAAA 644CfaaauuuaaaaL96 AD-74768 csasuccaAfcAfGf 563usCfsuuuCfuGfAfaucuGfuUfggaugsgsa 604 UCCAUCCAACAGAUUCAGAAAGC 645AfuucagaaagaL96 AD-74769 asasaaucAfaGf 564asAfscauAfgCfAfaaucUfuGfauuuusgsg 605 CCAAAAUCAAGAUUUGCUAUGUU 646AfUfuugcuauguuL96 AD-74770 asgsagcaAfaAfUf 565usUfscugGfcUfUfagauUfuUfgcucususg 606 CAAGAGCAAAAUCUAAGCCAGAG 647CfuaagccagaaL96 AD-74771 asuscauaUfgAfGf 566usGfsugaUfaUfUfagcuCfaUfaugausgsc 607 GCAUCAUAUGAGCUAAUAUCACA 648CfuaauaucacaL96 AD-74772 asasuaaaCfcUfCf 567usAfsacuUfgUfUfacgaGfgUfuuauususc 608 GAAAUAAACCUCGUAACAAGUUA 649GfuaacaaguuaL96 AD-74773 csasacagCfaUf 568usUfsuuaUfuUfGfacuaUfgCfuguugsgsu 609 ACCAACAGCAUAGUCAAAUAAAA 650AfGfucaaauaaaaL96 AD-74774 asasaacaAfcCfUf 569asUfsuuaCfcAfUfuuagGfuUfguuuuscsu 610 AGAAAACAACCUAAAUGGUAAAU 651AfaaugguaaauL96 AD-74775 csascuuaAfaAfCf 570usUfsucuAfcAfAfaaguUfuUfaagugsasa 611 UUCACUUAAAACUUUUGUAGAAA 652UfuuuguagaaaL96 AD-74776 asusuaaaAfuAfAf 571asGfsacaGfcGfAfacuuAfuUfuuaausasc 612 GUAUUAAAAUAAGUUCGCUGUCU 653GfuucgcugucuL96 AD-74777 csasaaacUfuCfAf 572usCfsacgUfuUfCfauugAfaGfuuuugsusg 613 CACAAAACUUCAAUGAAACGUGG 654AfugaaacgugaL96 AD-74778 asgsauggAfuCfAf 573usUfsgaaGfuUfUfugugAfuCfcaucusasu 614 AUAGAUGGAUCACAAAACUUCAA 655CfaaaacuucaaL96 AD-74779 csascuuaAfuAfCf 574usUfsguuUfuCfAfuaguAfuUfaagugsusu 615 AACACUUAAUACUAUGAAAACAA 656UfaugaaaacaaL96 AD-74780 ascsuaagUfcAfCf 575usUfsaaaGfuCfAfauguGfaCfuuagusasg 616 CUACUAAGUCACAUUGACUUUAA 657AfuugacuuuaaL96 AD-74781 usasuuaaAfuAfAf 576asUfsuuaGfaAfAfaguuAfuUfuaauasasg 617 CUUAUUAAAUAACUUUUCUAAAU 658CfuuuucuaaauL96 AD-74782 ascsacuuAfaUfAf 577usGfsuuuUfcAfUfaguaUfuAfagugususa 618 UAACACUUAAUACUAUGAAAACA 659CfuaugaaaacaL96 AD-74783 ususuaugAfaAfCf 578usGfscuuCfaUfUfagguUfuCfauaaasusa 619 UAUUUAUGAAACCUAAUGAAGCA 660CfuaaugaagcaL96 AD-74784 csasaacaUfuAfUf 579usAfsauaUfuCfAfauauAfaUfguuugsusu 620 AACAAACAUUAUAUUGAAUAUUC 661AfuugaauauuaL96 AD-74785 asasaccaGfuGfAf 580usUfsucuUfuGfAfuuucAfcUfgguuusgsc 621 GCAAACCAGUGAAAUCAAAGAAG 662AfaucaaagaaaL96 AD-74786 gsasguuaAfaGfUf 581usGfsgaaAfuAfUfaaacUfuUfaacucsgsa 622 UCGAGUUAAAGUUUAUAUUUCCC 663UfuauauuuccaL96 AD-74787 cscsaauaUfaAfAf 582usGfsguuUfaAfUfuguuUfaUfauuggsusc 623 GACCAAUAUAAACAAUUAAACCA 664CfaauuaaaccaL96 AD-74788 gsusggagAfaAfAf 583usAfsuuuAfgGfUfuguuUfuCfuccacsasc 624 GUGUGGAGAAAACAACCUAAAUG 665CfaaccuaaauaL96 AD-74789 asascucaAfcUfCf 584usUfsucaAfgUfUfuugaGfuUfgaguuscsa 625 UGAACUCAACUCAAAACUUGAAA 666AfaaacuugaaaL96 AD-74790 asasuguuCfaCfAf 585asGfsgagCfuUfAfauugUfgAfacauususu 626 AAAAUGUUCACAAUUAAGCUCCU 667AfuuaagcuccuL96 AD-74791 gscsagaaUfuAfAf 586usAfsauaCfaGfUfauuuAfaUfucugcsusu 627 AAGCAGAAUUAAAUACUGUAUUA 668AfuacuguauuaL96 AD-74792 usgsaaugAfaAfUf 587usUfsacaUfuUfCfuuauUfuCfauucasasc 628 GUUGAAUGAAAUAAGAAAUGUAA 669AfagaaauguaaL96 AD-74793 usascauaUfaAfAf 588usUfsgacUfuGfUfaguuUfaUfauguasgsu 629 ACUACAUAUAAACUACAAGUCAA 670CfuacaagucaaL96

TABLE 15B Additional Modified ANGPTL3 RNAi Agents. Sense SEQ SEQ SEQDuplex Sequence ID ID mRNA Target Sequence ID Name 5′ to 3′ NO:Antisense Sequence 5′ to 3′ NO: in NM_014495.3 NO: AD-66916asascuaaCfuAf 548 usUfsugaAfuUfAfaguuAfgUfuaguusgsc 589GCAACUAACUAACUUAAUUCAAA 630 AfCfuuaauucaaa AD-66922 gsasauauGfuCf 549usUfsgagUfuCfAfagugAfcAfuauucsusu 590 AAGAAUAUGUCACUUGAACUCAA 631AfCfuugaacucaa AD-74755 asasacucUfaAf 550asUfsuuaGfuCfAfaguuUfaGfaguuususa 591 UAAAACUCUAAACUUGACUAAAU 632AfCfuugacuaaau AD-74756 asascuaaCfuUf 551usGfsauuUfuGfAfauuaAfgUfuaguusasg 592 CUAACUAACUUAAUUCAAAAUCA 633AfAfuucaaaauca AD-74757 cscsagaaGfuAf 552usUfsuaaGfuGfAfaguuAfcUfucuggsgsu 593 ACCCAGAAGUAACUUCACUUAAA 634AfCfuucacuuaaa AD-74758 gsasacucAfaCf 553usUfscaaGfuUfUfugagUfuGfaguucsasa 594 UUGAACUCAACUCAAAACUUGAA 635UfCfaaaacuugaa AD-74759 csasacucAfaAf 554asGfsgcuUfuCfAfaguuUfuGfaguugsasg 595 CUCAACUCAAAACUUGAAAGCCU 636AfCfuugaaagccu AD-74760 ususccacGfuUf 555usCfsaauUfuCfAfagcaAfcGfuggaascsu 596 AGUUCCACGUUGCUUGAAAUUGA 637GfCfuugaaauuga AD-74761 gsusugcuUfgAf 556usGfsauuUfuCfAfauuuCfaAfgcaacsgsu 597 ACGUUGCUUGAAAUUGAAAAUCA 638AfAfuugaaaauca AD-74762 gsasucacAfaAf 557usUfsucaUfuGfAfaguuUfuGfugaucscsa 598 UGGAUCACAAAACUUCAAUGAAA 639AfCfuucaaugaaa AD-74763 uscsaagaUfuUf 558usUfscuaAfcAfUfagcaAfaUfcuugasusu 599 AAUCAAGAUUUGCUAUGUUAGAC 640GfCfuauguuagaa AD-74764 csasaaauCfaAf 559asCfsauaGfcAfAfaucuUfgAfuuuugsgsc 600 GCCAAAAUCAAGAUUUGCUAUGU 641GfAfuuugcuaugu AD-74765 gsasacuaCfuCf 560asCfsugaAfgAfAfagggAfgUfaguucsusu 601 AAGAACUACUCCCUUUCUUCAGU 642CfCfuuucuucagu AD-74766 asgsaaauUfuCf 561usUfsggaAfgAfUfagagAfaAfuuucusgsu 602 ACAGAAAUUUCUCUAUCUUCCAA 643UfCfuaucuuccaa AD-74767 usgsaacuGfaGf 562usUfsuuaAfaUfUfugccUfcAfguucasusu 603 AAUGAACUGAGGCAAAUUUAAAA 644GfCfaaauuuaaaa AD-74768 csasuccaAfcAf 563usCfsuuuCfuGfAfaucuGfuUfggaugsgsa 604 UCCAUCCAACAGAUUCAGAAAGC 645GfAfuucagaaaga AD-74769 asasaaucAfaGf 564asAfscauAfgCfAfaaucUfuGfauuuusgsg 605 CCAAAAUCAAGAUUUGCUAUGUU 646AfUfuugcuauguu AD-74770 asgsagcaAfaAf 565usUfscugGfcUfUfagauUfuUfgcucususg 606 CAAGAGCAAAAUCUAAGCCAGAG 647UfCfuaagccagaa AD-74771 asuscauaUfgAf 566usGfsugaUfaUfUfagcuCfaUfaugausgsc 607 GCAUCAUAUGAGCUAAUAUCACA 648GfCfuaauaucaca AD-74772 asasuaaaCfcUf 567usAfsacuUfgUfUfacgaGfgUfuuauususc 608 GAAAUAAACCUCGUAACAAGUUA 649CfGfuaacaaguua AD-74773 csasacagCfaUf 568usUfsuuaUfuUfGfacuaUfgCfuguugsgsu 609 ACCAACAGCAUAGUCAAAUAAAA 650AfGfucaaauaaaa AD-74774 asasaacaAfcCf 569asUfsuuaCfcAfUfuuagGfuUfguuuuscsu 610 AGAAAACAACCUAAAUGGUAAAU 651UfAfaaugguaaau AD-74775 csascuuaAfaAf 570usUfsucuAfcAfAfaaguUfuUfaagugsasa 611 UUCACUUAAAACUUUUGUAGAAA 652CfUfuuuguagaaa AD-74776 asusuaaaAfuAf 571asGfsacaGfcGfAfacuuAfuUfuuaausasc 612 GUAUUAAAAUAAGUUCGCUGUCU 653AfGfuucgcugucu AD-74777 csasaaacUfuCf 572usCfsacgUfuUfCfauugAfaGfuuuugsusg 613 CACAAAACUUCAAUGAAACGUGG 654AfAfugaaacguga AD-74778 asgsauggAfuCf 573usUfsgaaGfuUfUfugugAfuCfcaucusasu 614 AUAGAUGGAUCACAAAACUUCAA 655AfCfaaaacuucaa AD-74779 csascuuaAfuAf 574usUfsguuUfuCfAfuaguAfuUfaagugsusu 615 AACACUUAAUACUAUGAAAACAA 656CfUfaugaaaacaa AD-74780 ascsuaagUfcAf 575usUfsaaaGfuCfAfauguGfaCfuuagusasg 616 CUACUAAGUCACAUUGACUUUAA 657CfAfuugacuuuaa AD-74781 usasuuaaAfuAf 576asUfsuuaGfaAfAfaguuAfuUfuaauasasg 617 CUUAUUAAAUAACUUUUCUAAAU 658AfCfuuuucuaaau AD-74782 ascsacuuAfaUf 577usGfsuuuUfcAfUfaguaUfuAfagugususa 618 UAACACUUAAUACUAUGAAAACA 659AfCfuaugaaaaca AD-74783 ususuaugAfaAf 578usGfscuuCfaUfUfagguUfuCfauaaasusa 619 UAUUUAUGAAACCUAAUGAAGCA 660CfCfuaaugaagca AD-74784 csasaacaUfuAf 579usAfsauaUfuCfAfauauAfaUfguuugsusu 620 AACAAACAUUAUAUUGAAUAUUC 661UfAfuugaauauua AD-74785 asasaccaGfuGf 580usUfsucuUfuGfAfuuucAfcUfgguuusgsc 621 GCAAACCAGUGAAAUCAAAGAAG 662AfAfaucaaagaaa AD-74786 gsasguuaAfaGf 581usGfsgaaAfuAfUfaaacUfuUfaacucsgsa 622 UCGAGUUAAAGUUUAUAUUUCCC 663UfUfuauauuucca AD-74787 cscsaauaUfaAf 582usGfsguuUfaAfUfuguuUfaUfauuggsusc 623 GACCAAUAUAAACAAUUAAACCA 664AfCfaauuaaacca AD-74788 gsusggagAfaAf 583usAfsuuuAfgGfUfuguuUfuCfuccacsasc 624 GUGUGGAGAAAACAACCUAAAUG 665AfCfaaccuaaaua AD-74789 asascucaAfcUf 584usUfsucaAfgUfUfuugaGfuUfgaguuscsa 625 UGAACUCAACUCAAAACUUGAAA 666CfAfaaacuugaaa AD-74790 asasuguuCfaCf 585asGfsgagCfuUfAfauugUfgAfacauususu 626 AAAAUGUUCACAAUUAAGCUCCU 667AfAfuuaagcuccu AD-74791 gscsagaaUfuAf 586usAfsauaCfaGfUfauuuAfaUfucugcsusu 627 AAGCAGAAUUAAAUACUGUAUUA 668AfAfuacuguauua AD-74792 usgsaaugAfaAf 587usUfsacaUfuUfCfuuauUfuCfauucasasc 628 GUUGAAUGAAAUAAGAAAUGUAA 669UfAfagaaauguaa AD-74793 usascauaUfaAf 588usUfsgacUfuGfUfaguuUfaUfauguasgsu 629 ACUACAUAUAAACUACAAGUCAA 670AfCfuacaagucaa

TABLE 16 AngPTL3 Single Dose Screen in Hep3B cells and PrimaryCynomolgus Monkey Hepatocytes. Data are expressed as percent messageremaining relative to AD-1955 non-targeting control. Hep3b CynomolgusMonkey Hepatocytes 10 10 0.1 0.1 10 10 0.1 0.1 DuplexID nM_AVG nM_STDEVnM_AVG nM_STDEV nM_AVG nM_STDEV nM_AVG nM_STDEV AD-66916 5.2 2.1 17.94.1 5.2 0.4 40.5 5.4 AD-66922 12.5 2.1 41.5 17.0 4.6 0.6 43.5 8.2AD-74755 18.2 5.9 44.3 3.5 68.7 7.8 79.2 11.5 AD-74756 10.4 4.1 56.912.5 9.9 2.4 50.7 7.4 AD-74757 10.7 6.5 38.3 12.4 5.6 0.8 34.5 4.0AD-74758 19.3 3.1 120.4 19.5 24.1 6.9 78.4 17.7 AD-74759 20.4 3.9 132.134.5 26.7 4.0 80.4 8.3 AD-74760 21.6 4.7 140.1 16.8 12.8 1.5 72.5 6.4AD-74761 19.9 2.4 107.8 30.0 12.6 1.1 71.9 12.3 AD-74762 11.0 5.0 55.011.0 6.4 1.2 78.5 17.1 AD-74763 13.5 4.1 67.5 13.6 21.2 5.2 76.8 13.4AD-74764 10.4 3.0 70.7 21.4 15.8 1.4 63.1 4.8 AD-74765 16.5 3.6 83.116.4 22.0 2.5 82.2 10.4 AD-74766 11.3 6.2 56.0 25.3 7.8 0.4 51.3 2.1AD-74767 8.6 3.2 70.9 19.7 14.8 5.8 61.5 4.7 AD-74768 28.8 10.1 120.324.3 37.6 5.3 81.1 9.6 AD-74769 14.5 5.5 65.4 28.5 10.3 1.6 63.5 8.9AD-74770 42.4 13.0 95.0 23.6 59.8 8.1 104.6 14.8 AD-74771 30.0 12.2 79.715.3 73.7 10.0 87.6 17.1 AD-74772 22.9 3.8 60.4 10.9 80.9 9.1 83.8 8.0AD-74773 11.2 3.3 47.4 14.1 9.7 1.8 56.3 1.8 AD-74774 57.3 4.8 99.3 8.165.0 4.2 81.3 9.3 AD-74775 13.8 3.6 83.1 21.8 54.2 4.0 92.0 5.5 AD-7477650.6 11.5 114.3 39.7 80.2 11.0 97.3 9.4 AD-74777 14.8 3.8 76.7 27.0 39.412.1 92.1 4.2 AD-74778 22.0 13.0 62.8 24.6 15.6 3.0 70.4 7.4 AD-7477945.5 9.0 64.7 4.7 82.8 15.1 87.0 9.0 AD-74780 38.3 13.9 81.0 22.9 77.47.4 82.0 4.3 AD-74781 44.5 10.1 78.5 15.9 79.5 3.3 88.7 7.9 AD-7478259.1 18.5 137.3 65.4 79.1 3.6 88.5 4.3 AD-74783 115.4 42.4 109.6 32.691.8 5.6 95.3 9.2 AD-74784 11.5 4.6 47.8 18.3 8.6 2.8 56.7 10.7 AD-7478515.0 13.9 43.9 7.4 12.1 2.6 72.4 14.0 AD-74786 32.1 10.3 61.0 14.6 85.57.2 93.3 22.2 AD-74787 60.9 12.4 86.4 26.2 88.1 11.9 96.6 21.4 AD-7478817.8 7.7 69.9 23.8 19.3 1.2 79.2 15.3 AD-74789 30.6 3.6 64.0 6.9 24.61.3 76.0 9.5 AD-74790 15.4 3.5 56.9 10.5 9.2 0.6 58.7 12.4 AD-74791 30.76.3 49.3 6.9 81.3 8.3 87.3 3.8 AD-74792 10.5 2.4 30.1 6.4 10.5 1.2 54.77.3 AD-74793 12.1 4.9 30.9 16.2 13.2 2.1 83.2 12.7

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expressionof angiopoietin-like 3 (ANGPTL3), wherein said dsRNA comprises a sensestrand and an antisense strand, the antisense strand comprising a regionof complementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensenucleotide sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5, 7A,7B, 7C, 8A, 8B, 10A, 10B, 13A, 13B, 14, 15A, and 15B.
 2. (canceled) 3.The dsRNA of claim 1, wherein no more than four of the nucleotides ofthe sense strand are unmodified nucleotides; wherein no more than six ofthe nucleotides of the antisense strand are unmodified nucleotides;wherein all of the nucleotides of the sense strand are modifiednucleotides; or wherein all of the nucleotides of the antisense strandare modified nucleotides.
 4. (canceled)
 5. The dsRNA of claim 1, whereinthe region of complementarity is at least 17 nucleotides in length;19-23 nucleotides in length; or 19 nucleotides in length.
 6. The dsRNAof claim 1, wherein each strand is no more than 30 nucleotides inlength.
 7. The dsRNA of claim 1, wherein at least one strand comprises a3′ overhang of at least 1 nucleotide; or at least 2 nucleotides.
 8. ThedsRNA of claim 1, further comprising a ligand.
 9. The dsRNA of claim 8,wherein the ligand is conjugated to the 3′ end of the sense strand ofthe dsRNA.
 10. The dsRNA of claim 9, wherein the ligand is anN-acetylgalactosamine (GalNAc) derivative.
 11. The dsRNA of claim 10,wherein the ligand is


12. A double-stranded ribonucleic acid (dsRNA) for inhibiting expressionof angiopoietin-like 3 (ANGPTL3) in a cell, wherein said dsRNA comprisesa sense strand complementary to an antisense strand, wherein saidantisense strand comprises a region complementary to SEQ ID NO:2,wherein each strand is about 14 to about 30 nucleotides in length, andwherein said double-stranded RNAi agent is represented by formula(IIIe): (IIIe) sense: 5′-Na-Y Y Y-Nb-3′ antisense:3′-np′-Na′-Y′Y′Y′-Nb′-5′

wherein: np′ is a 2 nucleotide overhang and each nucleotide within np′is linked to a neighboring nucleotide via a phosphorothioate linkage;each Na, Nb, Na′ and Nb′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides; YYY and Y′Y′Y′ each independentlyrepresent one motif of three identical modifications on threeconsecutive nucleotides; wherein the sense strand comprises no more than4 2′-fluoro modifications; wherein the antisense strand comprises nomore than 6 2′-fluoro modifications; wherein the sense strand and theantisense strand each independently comprise two phosphorothioatelinkages at the 5′-terminus; and wherein the sense strand is conjugatedto at least one ligand.
 13. The dsRNA of claim 12, wherein the YYY motifoccurs at or near the cleavage site of the sense strand.
 14. The dsRNAof claim 12, wherein the double-stranded region is 15-30 nucleotidepairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotidepairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotidepairs in length; or 21-23 nucleotide pairs in length.
 15. The dsRNA ofclaim 12, wherein each strand has 15-30 nucleotides; or 19-30nucleotides.
 16. The dsRNA of claim 12, wherein the sense strandcomprises no more than 3 2′-fluoro modifications; or no more than 22′-fluoro modifications; or wherein the antisense strand comprises nomore than 5 2′-fluoro modifications; no more than 4 2′-fluoromodifications; no more than 3 2′-fluoro modifications; or no more than 22′-fluoro modifications.
 17. (canceled)
 18. The dsRNA of claim 12,wherein the ligand is one or more GalNAc derivatives attached through amonovalent, a bivalent, or a trivalent branched linker.
 19. The dsRNA ofclaim 12, wherein the ligand is


20. The dsRNA of claim 12, wherein the ligand is attached to the 3′ endof the sense strand.
 21. (canceled)
 22. A cell containing the dsRNA ofclaim
 1. 23. A pharmaceutical composition for inhibiting expression ofion of an ANGPTL3 gene comprising the dsRNA of claim
 1. 24. A method ofinhibiting ANGPTL3 expression in a cell, the method comprising: (a)contacting the cell with the dsRNA of claim 1; and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression ofthe ANGPTL3 gene in the cell. 25.-27. (canceled)
 28. A method oftreating a subject having a disorder that would benefit from a reductionin ANGPTL3 expression, comprising administering to the subject atherapeutically effective amount of the dsRNA of claim 1, therebytreating said subject.